Identification of a Member of
Enterobacteriaceae Using
Physiological Factors
Dalton Vanadis. Section 2; Bench 4 [Submitted: November 09, 2011] unk #2
Introduction:
The goal of this experiment is the identification of
an existing member of Enterobacteriaceae, which requires it
to be eliminated from related species and genera including
such groups as Shigella, Salmonella, Citrobacter, Klebsiella,
Enterobacter, Serratia, Proteus, Providencia, Yersinia,
Pantoea, and other assorted individual representative species.
Primary properties of this family include a negative result
from Gram staining, facultative anaerobic respiration, a rod
morphology, and sugar fermentation. These properties are
representative of the family, various other properties within
the family can be used to identify individual genera and
species.
Identification was based on physiological factors
such as differential sugar fermentation, decarboxylase
activity, cysteine desulfurase activity, cell motility, use of the
TCA cycle to metabolize exogenous citrate, and
tryptophanase activity which are important differentiating
factors for the individual members of Enterobacteriaceae.
Initially, main identifying factors were confirmed
for the sample, (gram-stain response and overall cell and
colony morphology), then purity of the sample was assessed
by streaking, isolated colonies were then used in a Trypticase
Soy Agar (TSA) inoculation to provide a solid media source
of for further testing. After these confirmatory procedures
and preparative methodologies were completed, an initial
round of testing was conducted, focusing on glucose
fermentation, indole production, H2S production, citrate
utilization, and motility. These tests were used to exclude
several genera, the remaining possibilities were then used to
determine subsequent tests for a final identification. These
focused on carboxylase activity with two amino acids as well
as a test of raffinose fermentation capability.
Based upon the results observed from the tests
conducted, it was determined that Serratia marcescens was
the member of Enterobacteriaceae present.
Materials and Methods:
Confirmation and Isolation of Enterobacteriaceae1:
The original sample was gram stained initially to
confirm general cell morphology and reaction to the gram
stain itself. The sample was then streaked on a TSA plate for
isolation and allowed to incubate for approximately 24 hours.
Macro-morphology of isolated colonies was used to visually
confirm reasonable purity of the sample. Select colonies were
then used for a second confirmatory gram stain and for
inoculation of a TSA slant for use as a pure sample to
perform further identification tests with.
Initial Physiological Tests1
These tests included: A Bromcresol Purple (BCP)
glucose test with a Durham tube to measure acidic
byproducts from glucose fermentation and gas production
therein. A BCP broth containing glucose and the Durham
tube was inoculated with the sample and incubated for 48
hours and then evaluated based on gas presence and
coloration of the solution.
A Methyl Red-Voges-Proskauer (MR-VP) assay for
mixed acid fermentation and 2,3-butanediol production. This
test was conducted using a single initial growth medium of
MR-VP broth inoculated with sample and incubated for 48
hours. It was then split into two sections which were tested
using the addition of the indicator methyl red in one to
measure any formation of acetate, succinate, or formate upon
glucose fermentation. The other aliquot was used to measure
acetoin formation with Barritt’s reagent.
A citrate utilization test with a Simmon’s Citrate
slant to subjectively evaluate the usage of citrate as a carbon
source. Some of the sample was used to inoculate the slant
which was incubated for 48 hours and then evaluated based
on changes in the coloration of the medium due to alkalinity
changes associated with the use of citrate as a carbon source.
A tryptophanase assay (measuring indole
production) using a tryptone broth as the growth medium and
Kovac’s reagent as the indicator to evaluate the presence of
indole as a byproduct of the deamination of tryptophan. The
medium was incubated for 48 hours after being inoculated
with sample. After incubation the reagent was added and
color changes were noted to determine the results of the test.
A Kligler’s Iron Agar (KIA) test to measure H2S
production from cysteine desulfurase activity. Some of the
sample was stabbed through a KIA slant and swabbed along
the surface. This would allow confirmation of glucose and/or
lactose fermentation by the sample as well as indicate
through color change presence of H2S. The medium was
incubated for 24 hours and then evaluated immediately.
And finally a motility assay using a stab of soft agar.
A simple stab of the sample through a tube of soft agar was
incubated for 48 hours and then evaluated based on any
noticeable movement of sample through the medium.
Secondary/Confirmatory Physiological Tests:
These tests included: A BCP Raffinose test to
evaluate the production of acidic byproducts resulting from
raffinose fermentation. This was conducted by inoculating a
BCP solution containing raffinose with the sample,
incubating for 48 hours and evaluating changes in the
coloration of the BCP indicator.
A lysine decarboxylase test to evaluate the presence
of the eponymous enzyme. This test was conducted using
Moeller’s decarboxylase broth containing lysine inoculated
with some of the sample. A layer of sterile mineral oil was
used to produce an anaerobic environment. The solution was
incubated for approximately 48 hours and the final coloration
of the solution as compared to a control containing no amino
acid addition was assessed for decarboxylase activity.
An ornithine decarboxylase test, again to evaluate
the presence of the eponymous enzyme. The same procedure
used in the lysine decarboxylase test was used here, with
ornithine replaced as the added amino acid in the Moeller’s
decarboxylase broth.
Results:
BCP glucose gas production:
This test yielded a positive result for acidic
byproducts while producing a negative result for gas
production. A positive and negative control was conducted
for this test and their results were as expected, supporting the
validity of these results.
Acetate, succinate, and formate production from glucose
fermentation using methyl red as an indicator:
This test was negative for any of the relatively
strong acidic products acetate, succinate, and formate as
evidenced by the yellow color of the solution. A positive and
negative control was conducted for this test and their results
were as expected, supporting the validity of these results.
Voges-Proskauer detection of acetoin (intermediate in 2,3butanediol formation from glucose fermentation):
This test displayed a positive result for the mildly
acidic product, acetoin as evidenced by a pinkish-red
solution. A positive and negative control was conducted for
this test and their results were as expected, supporting the
validity of these results.
Figure 1: KIA Slant results after 24 hour incubation. Unknown bacteria (S.
marcescens)
Utilization of exogenous citrate evidenced by increased
alkalinity in the external medium:
The result for this test was positive as evidenced by
the blue hue taken on by the bromothymol blue indicator in
response to a higher alkalinity in the medium. A positive and
negative control was conducted for this test and their results
were as expected, supporting the validity of these results.
Production of indole by tryptophanase measured via
Kovac’s Reagent:
This test displayed a negative result, with the
solution taking on a yellow hue indicative of a lack of
presence of indole as a byproduct of tryptophanase activity.
A positive and negative control was conducted for this test
and their results were as expected, supporting the validity of
these results.
Assay for cysteine desulfurase activity coupled with
confirmatory results of glucose fermentation, lactose
fermentation, and gas formation from glucose
fermentation:
The results indicated from using a KIA slant
include: Negative for cysteine desulfurase activity (no H2S
production), positive for glucose fermentation, positive for
lactose fermentation, negative for gas formation from glucose
fermentation. This can be seen in Figures 1 and 2
demonstrating the result of the unknown assay as well as the
E. coli control. In addition to E. coli a positive control for
H2S production was conducted for this test all control results
were as expected.
Motility of both the control and unknown could not
be reliably determined due to human error and difficulties
inherent in interpreting the results of this assay.
Figure 2: KIA Slant results after 24 hour incubation. E. coli control
Figure 3: BCP Raffinose solution results (S. marcescens), 48 hour incubation
Acidic product formation from fermentation of raffinose
as evidenced by color change in BCP solution:
This test produced a negative result, with no visible
color change that would have resulted had fermentation
occurred. This can be seen in Figure 3.
Presence of lysine and ornithine carboxylases as
evidenced by alkaline shifts within the medium:
Both lysine and ornithine carboxylase tests were
positive for enzyme activity and presence for the unknown
this is evidenced by a purple solution color. This can be seen
in Figures 4 and 5. Furthermore, a control was conducted
with no amino acid present in the Moeller’s decarboxylase
broth, it’s results were as expected with the production of a
yellow acidic solution. This can be seen in Figure 6.
Figure 4: Lysine decarboxylase test (S. marcescens), positive for carboxylase
activity
Figure 5: Ornithine decarboxylase test (S. marcescens), positive for
carboxylase activity
Figure 6: No amino acid decarboxylase control (S. marcescens), negative for
carboxylase activity, acidic solution from glucose fermentation
Discussion:
The original physiological tests conducted narrowed
down the candidate pool from thirty-eight possibilities to six.
This exclusionary methodology was applied to all results
obtained and accepted as usable.
The lack of gas production from glucose allowed for
the effective elimination of the Citrobacter and Enterobacter
genera from further consideration, as well as the
representative species Escherichia coli, Edwardsiella tarda,
Hafnia alvei, and Rahnella aquatilis. Along with multiple
members of the genera Salmonella, Klebsiella, and Proteus
as well. As all of these species and genera would produce
gas to a noticeable extent. The lack thereof is a way to
numerically reduce the candidate pool.
The methyl red test effectively removed the genera
Shigella, Salmonella, Proteus, Providencia, and Yersinia
from consideration as all of these genera would have
provided unequivocal positive results when subjected to a
methyl red test for mixed acid production. Furthermore the
genus Citrobacter was confirmed to not be a member of the
candidate pool by this test. This test left some representative
species as possible candidates, mostly these were focused in
the genera Serratia and Pantoea.
The Voges-Proskauer test for mixed acid production
yielded a positive result that led to the effective elimination
(and in some cases confirmation of elimination) from
consideration of the Shigella, Salmonella, Citrobacter, and
Providencia genera from potential candidates for the identity
of the unknown sample. Along with such species as E. coli,
Edwardsiella tarda, P. vulgaris, and several Yersinia species.
The citrate utilization test, with a positive result of
exogenous use of citrate as a carbon source allowed for the
confirmatory exclusion of Yersinia, Shigella, and E. coli from
consideration as potential unknown candidates.
The indole test did little in terms of narrowing the
candidate pool, however it did confirm that Providencia as a
genus is unlikely to be a candidate for the unknown alongside
several individual species scattered through the genera of
Enterobacteriaceae.
The KIA slant’s negative result for cysteine
desulfurase activity is indicative of Salmonella, and Proteus
not being potential genus candidates for the unknown,
alongside several assorted individual species.
Overall, these preliminary physiological results led
to five potential candidates: Serratia marcescens, S.
liquifaciens, S. rubidaea, Pantoea agglomerans, and P.
disperse. These candidates were determined based on the fact
that they contained the highest number of points of agreement
in terms of physiological test results, with all six tests utilized
matching expected properties of these candidates.
The genera Serratia and Pantoea can be separately
identified and/or excluded as a whole using a lysine
decarboxylase test. The results of this test indicated that
Pantoea is not a valid candidate for the identity of the
unknown sample.
The species of Serratia could be individually
separated and identified by using an ornithine decarboxylase
test and a BCP raffinose test in conjunction with each other.
The results of these two tests when combined would
effectively provide enough granularity in the results to give a
most likely candidate. The ornithine decarboxylase test
effectively exclude S. rubidaea while the BCP raffinose test
effectively excludes both S. liquifaciens and S. rubidaea as
potential candidates. This leaves S. marcescens as the only
candidate within the accepted list of Enterobacteriaceae
members to have met the physiological criteria matching all
nine tests conducted.
Serratia marcescens has previously been shown to
be negative for indole production.2 It has also been shown
that the MR-VP test produces reliable, valid, and
reproducible results with a negative Methyl Red and a
positive Voges-Proskauer result.3 S. marcescens has also
been shown to have a positive result on a Simmon’s citrate
slant4, which lends further credence to the results produced
here. Furthermore, the lack of production of gas from glucose
fermentation in S. marcescens has been previously
documented as well.5 Finally, with regard to the preliminary
physiological tests conducted and their confirmed relevance
in support of S. marcescens as the unknown bacteria, a
negative result for hydrogen sulfide production (measured via
KIA slant) has been previously documented as well.6 All of
these factors support S. marcescens as a likely candidate for
the unknown culture.
The second round of tests and their results are also
similarly supported in the literature (ornithine
decarboxylase2, lysine decarboxylase2, 7, BCP raffinose2, 8) all
results taken together are strongly indicative of S. marcescens
as the unknown organism present in the pure culture.
Further confirmation of the identity of this organism
could be pursued through several potential routes. One such
route may be through biochemical or molecular phylogenetic
methods such as confirming the 16S rRNA sequence against
known databases. In terms of physiological confirmation, this
could potentially be achieved through further sugar
fermentation tests in BCP mediums as well as an arginine
decarboxylase test, phenylalanine deaminase or malonate
utilization tests would help definitively confirm the identity
of the organism using known characteristics.
Overall, the only particular error that affected the
results of this classification was inconsistencies noted with
the motility test. However, this did not end up affecting the
overall classification and identification of the unknown
organism. The final result of S. marcescens still stands and is
supported by nine different physiological tests which are
widely accepted values for the identification of this organism.
References:
1. Hung Ko-Hsuan, A. 2011. Oral interviews regarding
experimental procedure.
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Martin, MD; R.E. Ritts Jr., MD; 1970. Serratia marcescens:
Biochemical Characteristics, Antibiotic Susceptibility
Patterns, and Clinical Significance JAMA.
1970;214(12):2157-2162. doi:10.1001/
jama.1970.03180120029006
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N-Acyl-L-Homoserine Lactone Quorum Sensing Controls
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