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Brenna Moya
Unknown: 21
21A: Alcaligenes faecalis
21B: Streptococcus pneumoniae

Introduction

Alcaligenes faecalis is a gram-negative bacillus with a flagella that belongs to the Alcaligenaceae
family. This nonfermentive aerobic bacteria is oxidase-positive, nonencapsulated, and is named for its ability to
produce an alkaline reaction (Hasan et al., 2019). Alcaligenes faecalis is commonly found in soil, water, and is
also part of the human intestinal flora. Though part of the normal human microbiota, Alcaligenes faecalis is an
opportunistic pathogen that when given the chance it can migrate to the respiratory track and or blood causing
infections (Momtaz et al., 2018). Examples of Alcaligenes faecalis-associated infections include chronic otitis,
abscesses, bloodstream infections, meningitis, endocarditis, urinary tract infections (UTIs), and peritonitis.
While Alcaligenes faecalis incidences are rare, when they do occur they are often severe. Alcaligenes faecalis is
pandrug-resistant, meaning that it is resistant to the majority of all commercially available antimicrobials
(Hasan et al., 2019). As of 2017, Alcaligenes faecalis isolates were resistant to seven out of ten of the
commercial antimicrobials that are commonly used as a treatment for UTI’s (Momtaz et al., 2018). Despite
being a multidrug-resistant bacteria, today it is extensively used in sewage treatment and in pharmaceutical
industries. Also, Alcaligenes faecalis is commonly utilized in environmental industries since some strains can
break down many organic contaminates in the biodegradation process of organic pollutants and hazardous
industrial waste-materials (Hasan et al., 2019). Overall, while having the ability to cause severe infections,
Alcaligenes faecalis rarely infects humans and is used to aid various industries.

Streptococcus pneumoniae is a gram-positive facultative anaerobe with a cocci morphology and a

strepto- (chain) grouping and the cause of community-acquired pneumonia (CAP). Infections caused by
Streptococcus pneumoniae are present all over the world and are typically prevalent during the winter and early
spring months (Dion and Ashurst, 2022). In 1881, Streptococcus pneumoniae was identified as a major
pathogen affecting the respiratory tract. Though despite the years of research and effective antimicrobials,
pneumococcal infections are still a formidable opponent of the healthcare field (Catterall, 1999). As of 2021,
community-acquired pneumonia (CAP) is the seventh leading cause of death and has an estimated
hospitalization cost of up to nine billion dollars each year in the Unites States. A thirty-day hospitalization with
a CAP has a mortality rate as high as 22% and is the leading cause of death between all infectious diseases
(Dion and Ashurst, 2022). Also, in third- world countries around five million children that are under the age of
five die each year due to respiratory pneumococcal infections (Catterall, 1999). The reason Streptococcus
pneumoniae is such a potent bacteria is due to its polysaccharide capsule and adhesion ability. The
polysaccharide capsule, which is the most important virulence factor, aids in the escape of phagocytosis by
preventing the entry of granulocytes to the cell wall. Streptococcus pneumoniae is also very virulent due to its
ability to adhere to the respiratory epithelium and cause a significant inflammatory response upon invasion.
Streptococcus pneumoniae has a totally of 92 different serotypes, and serotypes 6, 14, 18, 19, and 23 have been
identified as the common ones to cause infections. These serotypes are usually identified based on their
appearance on a culture growth medium. The type that appears transparent typically colonize the nasopharynx
and the type that appears opaque is usually present in the lungs, brain, and bloodstream (Dion and Ashurst,
2022). While Streptococcus pneumoniae has been around for quite so time, it still continues to be a major
problem all over the globe due to its potent virulence factors and abundant serotypes.
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Results and Discussion

Before the evaluation of the unknowns, dichotomous keys were created using the gram-positive and
gram-negative result sheets provided by instructor. These keys, which display how a known organisms would
react in each test, were first separated base on either being a gram-positive or gram-negative organism. Then
each key was separated further by choosing tests that could identify multiple characteristics of an organism.
Each test that was performed was checked after 24 hours and based on the results and aid from the dichotomous
keys, would either point to which test should be done next or the identification of the unknowns. These keys
ensured that tests were not only chosen logically, but based off of results the identification of unknowns were
easily decided, making sure no problems in the identification process occurred.

Gram-stain

Gram-Positive Gram-Negative
Bacillus megaterium Alcaligenes faecalis
Bacillus subtilis Citrobacter freundii
Enterococcus faecalis Escherichia coli
Micrococcus luteus Klebsiella aerogenes
Sporosarcina ureae Klebsella pneumoniae
Staphylococcus aureus Proteus mirabilis
Staphylococcus epidermidis Proteus vulgaris
Staphylococcus saprophyticus Pseudomnas aeruginosa
Streptococcus bovis Serratia marcescens
Streptococcus mutans Shigella flexneri
Streptococcus pneumoniae
Streptococcus pyogenes

SIM Test

Cocci
Enterococcus faecalis Strepto-
Micrococcus luteus Streptococcus bovis
Sporosarcina ureae Streptococcus mutans
Staphylococcus aureus Streptococcus “+” H2S (Black)
“-” H2S (No black color)
Staphylococcus epidermidis pneumoniae Citrobacter freundii
Staphylococcus saprophyticus Proteus mirabilis Alcaligenes faecalis
Streptococcus pyogenes
Streptococcus bovis Proteus vulgaris Escherichia coli
Streptococcus mutans Klebsiella aerogenes
Streptococcus pneumoniae Klebsella pneumoniae
Streptococcus pyogenes Pseudomnas aeruginosa
Serratia marcescens
Mannitol Test Shigella flexneri

MR Test
“-” Stays orange/red in color “+” Colors change to yellow
Streptococcus bovis Streptococcus bovis
Streptococcus pyogenes Streptococcus mutans
Streptococcus pneumoniae Streptococcus pyogenes

“+” Color change to red “-” Stays colorless

Escherichia coli Alcaligenes faecalis
Bile Esculin Test Klebsella pneumoniae Klebsiella aerogenes
Serratia marcescens Klebsella pneumoniae
Shigella flexneri Pseudomnas aeruginosa
Serratia marcescens

“-” Stays clear yellow

“+” Turns black/dark brown
Streptococcus pyogenes
Streptococcus bovis
Streptococcus pneumoniae
Oxidase Test

Bacitracin Resistance
Test
“+” Color change to purple “-” No color change
Alcaligenes faecalis Klebsiella aerogenes
Pseudomnas aeruginosa Klebsella pneumoniae
Serratia marcescens

“R” Growth right up to disc “S” Zone of inhibition

Streptococcus pneumoniae Streptococcus pyogenes
Phenylalanine Test

“-” No color change to purple

“+” Color change to green Pseudomnas aeruginosa
Alcaligenes faecalis
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The first test that was performed on the unknown bacterium was a gram-
stain that determined not only whether if the bacteria was a gram-
positive or gram-negative, but also determined morphology and Figure 2. Gram-positive, cocci shaped
grouping. Unknown 21A turned pink after the gram stain, which bacteria with a strepto- or chain
Figure 1. Gram-negative, bacillus or rod
indicated a gram-negative bacteria and had a bacillus or rod grouping
shapedFigure 5. Oxidase test result of
bacteria.
morphology (fig. 1). Unknow 21B was purple after the completed unknown 21A
gram-stain, which indicated a gram-positive bacteria and had a
cocci or sphere morphology with a strepto- or chain-like grouping
Figure 6. Phenylalanine
(fig. 2). The gram-stain that was performed narrowed down the test result for unknown
possible tests needed to be performed on each unknown to 21A
discover the identities.

The next test that was ran on unknown 21A was a

sulfide, indole, and motility test, also known as a SIM test. The
main result that this test identified that would aid further in
Figure 3. Sulfide, indole, narrowing down the identity of unknown 21A was the sulfide test.
and motility (SIM) test The SIM test results displayed that the bacteria of unknown 21A
results for unknown 21A. is not a sulfide producing bacteria since the medium did not turn
black in color, but rather stayed colorless (fig.3). This negative
test result narrowed down the potential bacteria identity to Alcaligenes faecalis, Escherichia
Figure 4. Methyl red coli, Klebsiella aerogenes, Klebsella pneumoniae, Pseudomnas aeruginosa, Serratia
(MR) test result of marcescens, and Shigella flexneri. These bacteria are all non-sulfide producing bacteria, so to
unknown 21A limited the options of the identity of the unknown, a methyl red or MR test was performed.

A MR test, which tests for heterolactic or mixed fermenters, was

performed next on unknown 21A. Methyl red reagent was added to the
broth after the incubation period. When the reagent is added the broth
will either turn red to indicate acid production due to fermentation or
have no visible colors change. After the addition of the methyl red
reagent to unknown 21A, it remained colorless and had no color change,
which indicates that unknown 21A is not heterolactic (fig. 4). This test
result narrowed the potential candidates of the identity of unknown 21A
to Alcaligenes faecalis, Klebsiella aerogenes, Klebsella pneumoniae,
Pseudomnas aeruginosa, and Serratia marcescens.

The next test that was done was an oxidase test, which tests for the
presence of the enzyme cytochrome c oxidase. The reagent TEMED or
tetramethyl-p-phenylenediamine is added to the bacteria and will either demonstrate a color
change to purple to indicate a positive test result or will not change to purple to show a
negative test result. Unknown 21A produced a positive result with the color change to
purple, which indicated that the unknown has the enzyme cytochrome c oxidase (fig. 5).
This positive test result narrowed down the identity of the unknown even further, leaving
Alcaligenes faecalis and Pseudomnas aeruginosa as the two candidates.

The final confirmation test that was ran was a phenylalanine test, which tests for the
presence of phenylalanine deaminase. The reagent ferric chloride was added to the growth
medium and if there was a color change to a dark green color it would indicate a positive
test result, if there was no color change this meant the test was negative. When the reagent
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was added to unknown 21A there was a dark green color change, which is not only a
positive test but also indicates that the unknow possesses the enzyme phenylalanine
deaminase (fig. 6). The result of this test determine the identity of unknown 21A to be
Alcaligenes faecalis.

After the gram-stain concluded that unknown 21B was a gram-positive bacteria
with a cocci morphology and a strepto- grouping, a mannitol test was chosen to be the first
test to be performed. A mannitol tests for fermentation of mannitol and will either produce
a color change from phenol red to yellow for a positive test result or will have no color
Figure 9. Bacitracin resistance test result for
change remaining an orange/red color for a negative test result. Unknown 21B had a
unknown 21 B
negative test result since it did not produce a color change, which indicates that the
unknown is a non-mannitol fermenter (fig. 7). This test narrowed down the potential
identity of unknown 21B to Streptococcus bovis, Streptococcus pyogenes, and
Streptococcus pneumoniae.
Figure 8. Bile esculin or
The next test to be performed was a bile esculin or BE test, which tests for a BE test result for
unknown 21B
microbe’s ability to hydrolyze esculin. A positive test result is indicated by a color change
to a black or dark brown color and a negative test result is determine by no color change.
When unknown 21B was inoculated into the BE medium and left to incubate it produced a
negative result by having no color change (fig. 8). The results of this negative test left the
possible identity of unknown 21B to be between two bacterium, Streptococcus pyogenes
and Streptococcus pneumoniae.

The final confirmation test that was performed was a

bacitracin resistance test, which tests a microbe’s
sensitivity to bacitracin. A “R” or resistant test result is
determined by the growth of the bacteria all the way up Figure 7. Mannitol test
to the bacitracin disc and a “S” or sensitive result is results for unknown 21B
displayed by a zone of inhibition or an area of no grow
around the bacitracin disc. Unknown 21B has growth
right up to the disc, which displays a “R” or resistant
test result (fig. 9). This confirmation test result
determined that the identity of unknown 21B to be Streptococcus
pneumoniae.

Conclusion
Overall with the aid of the dichotomous keys the process of determining the identity of both unknowns
went very smooth. A gram-stain was conducted first and 21A was determine to be a gram-negative with a
bacillus morphology and 21B was concluded to be a gram-positive with a cocci morphology with a strepto-
grouping. For unknown 21A a SIM test was ran first to determine sulfide production. The result of the sulfide
production was negative since the medium did not turn black. A MR or methyl test was the next test to be
perform on unknown 21A and it also had a negative result due it not changing colors after the methyl red
reagent was added. Then an oxidase test was done and when the unknown 21A bacteria was applied to the
reagent TEMED it did change to a purple color, which indicated a positive test result. The oxidase test narrowed
down the identity of the unknown to two potential bacterium, Alcaligenes faecalis and Pseudomnas aeruginosa.
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The unknown 21A confirmation test was a phenylalanine test , which did produce a positive result since there
was a color change to dark green after the ferric chloride reagent was added. This final test concluded that the
identity of unknown 21A to be Alcaligenes faecalis.

After the results of the gram-stain of unknown 21B, a mannitol test was the first test to be ran. Unknown
21B produced a negative test result since there was no color change of the broth from red to yellow. The next
test ran to narrow down the identity of unknown 21B was a BE test, which also had a negative test result since
the medium did not turn black or dark brown. The negative test result left only two potential identities for
unknown 21B, which were Streptococcus pyogenes and Streptococcus pneumoniae. A bacitracin resistance test
was chosen to be the final confirmation test and unknown 21B had a “R” or resistant test result since the
bacteria grew all the way up to the bacitracin disc. This confirmation test determined that the identity of
unknown 21B to be Streptococcus pneumoniae. The identification process of the unknowns did not encounter
any problems and all tests were chosen and read with confidence due to the creation of the dichotomous keys.
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References

Catterall, J. R. (1999). Lung infections bullet 5: Streptococcus pneumoniae. Thorax, 54(10), 929–937.
https://doi.org/10.1136/thx.54.10.929

Dion, C. F., & Ashurst, J. V. (2022). Streptococcus pneumoniae - statpearls - NCBI bookshelf. Retrieved May
1, 2022, from https://www.ncbi.nlm.nih.gov/books/NBK470537/

Hasan, M. J., Nizhu, L. N., & Rabbani, R. (2019, July 15). Bloodstream infection with Pandrug-resistant
alcaligenes faecalis treated with double-dose of Tigecycline. IDCases. Retrieved May 1, 2022, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656691/#:~:text=Alcaligenes%20faecalis%20is%20a
%20gram,in%20certain%20medium%20%5B1%5D.

Momtaz, F., Ali, M. H., Hossain, M. N., Foysal, M. J., Sumiya, M. K., & Islam, K. (2018). Characterisation of
multidrug-resistant alcaligenes faecalis strain AF1 isolated from patient of rutis: A study from
Bangladesh. JOURNAL OF CLINICAL AND DIAGNOSTIC RESEARCH.
https://doi.org/10.7860/jcdr/2018/37240.12179