Abstract

The bacterial family Enterobacteriaceae houses a vast variety of species each with its own set of
morphological, physiological and biological characteristics. This experiment the biotyping of the
bacterial species which we received was used to identify the species. A series of biochemical tests
were run such as an IMVIC test, an API tests etc. The biochemical test results were compared to
bacteria in Bergey’s Manual 2nd edition, volume 2 and it was indeed determined that the organism
in question was identified as Shigella sonnei. This research is of importance as S. sonnei is one of the
most common bacterial spices we as humans encounter; the more we know about these bacteria
the more we can understand them and help prevent diseases as well as death.

Introduction

The family Enterobacteriaceae belongs to the phylum Proteobacteria and the class of
Gammaproteobacteria which is the largest phylum in the domain of Bacteria. Bacteria are a group of
organisms with an immense range of morphological, biochemical and physiological properties. It is
comprised of facultative anaerobic bacteria as well as 80% of the bacteria are gram-negative which
are present predominantly in the hospitals of the United States of America (Karlowsky et al, 2003).
This family contains symbionts which are beneficial to the environment. The family
Enterobacteriaceae consists of harmful pathogens therefore when the species are grouped together
they are straightforwardly identifiable (Farmer et al, 1985).

The genus Shigella houses many harmful pathogens; with one of these pathogens being S. sonnei
which is the causative agent to the disease shigellosis. The gram-negative bacterium which causes
the diarrheal disease affects people from developing and developed countries (Liu et al, 1995). The
genus as a whole has also taken a massive stride into longer survival as they have developed
resistance to antibiotics such as ampicillin, streptomycin or tetracycline to mention a few (Pan et al,
2006). There is no vaccine yet known to the genus as the development is difficult as there is no
challenge dose of an active strain that can be used in connection to the infection of humans (Taylor
et al, 1993). This also serves an economic burden to a country as funds need to be set aside in order
to treat people and help prevent the disease.

Biochemical tests are an indicator media used for the identification of species that requires and
incubation of 24 hours or longer (Conway et al, 2001). Inoculation is a method used in a biochemical
test which allows for the identification of an organism after the period of incubation. A type of
biochemical testing are Analytical Profile Index (API) strip. It consists of 20E test kits that
distinguishes the different test elements and distinguishes the organisms based on the results it is
observed(Seo and Brackett, 2005). Genotypic methods have been created such as DNA probes which
are expensive and technically demanding (Conway et al, 2001). Alternative methods such as
polymerase chain reactions or DNA sequencing have been preferred by many as 16s rRNA for
various species are diverse within a genus therefore making the form of identification very
reliable(Woo et al, 2003).

A study of 20 biochemical Analytab systems has been tested in order to identify member of
Enterobacteriaceae with an accuracy of 90%. As the test was repeated several times the accuracy in
identification has increased irrespective of the time by 3%(Washingston et al, 1971). Another has
showed that the identification of the members have been present in meat products. This method of
identifaction was more preferable by others than the genotypic method (Stiles and NG, 1981). It has
been proven that this method is more accurate and cost effective in comparison to the genotypic
method.
The time constraints on biochemical testing can be seen as a negative aspect as results are often
required immediately. A study had showed that the biochemical test that was performed provided
results that did not contain accurate phenotypic information in order to identify the organism
(Drancourt et al, 2000). It was identified by researchers in other studies that biochemical tests do
not indicate any colonial origin (Gomez-De-Leon et al, 2000). Researcher would prefer the genotypic
method as more information of the organisms origin is identified.

Researches have favoured genetic methods as biochemical method do provide adequate

information in order to identify the organism(Lui et al, 1995). PCR, DNA restriction fragment length
polymorphisms and rRNA gene restriction patterns are genetic methods which provides complex
patterns and do not contain discriminatory capabilities (Gomez-De-Leon et al, 2000). A collection of
environmental and clinical uncultured mircoorganisms has led to many databases storing
information in order for sequencing of genotypes to occur (Dancourt et al, 2000).

The drawbacks of genetic method is that it is expensive as it requires equipment that is expensive.
Rearches do not have the funds available to perform this method therefore replying on the
alternative method of biochemical testing. The equipment used maybe complicated to operate but it
is cost-effective. In order for the equipment to operate workers are provided with training in order
the prevent damage to the equipment which could be more expensive

The aim of the is experiment is to identify the unknown organism through the use of the Biotyping
method. The experiment performed aids in the identification of organisms which are beneficial and
which are harmful to the environment and people. This aid in the ability to gain more information
about the characterisation of the organism studied.
Methods and Materials

The above mentioned biochemical tests were performed on unknown organism labelled number 35
in order to identify the type of bacterial organism by its reaction to various biochemical tests based
on its metabolic activities. The reagents were supplied by the company Bio Lab.

Carbohydrate Fermentation Test:

Peptone broths containing phenol red dextrose, lactose and sucrose in Durham tubes were
aseptically (by a Bunsen burner) spread with a loopful of the unknown bacterial culture. The
tubes were incubated for 48 hours at 37ºC.

Triple Sugar Iron (TSI) Agar Test:

Triple sugar iron agar slants containing the molecules glucose, sucrose and lactose were
streaked with the unknown bacterial culture which sterilely inoculated deeply by stabbing the
agar with a sterile inoculating needle. The TSI tubes were then incubated for 48 hours at 37ºC.

Starch Hydrolysis:

A starch agar plate was aseptically streaked in a straight line with the unknown bacterial culture
using a sterile inoculating loop. The plate was then incubated for 48 hours at 37ºC. After
incubation, several drops of Gram’s iodine was added to the streaked plate to turn the plate to
a blue/brown complex.

Proteolysis Activity and Enzyme Activity:

Hydrogen Sulfide Production and Motility:

Sulfide indole motility (SIM) agar deeps were aseptically inoculated with the unknown
bacterial culture using a sterile inoculating needle by stabbing the agar ¾ to the bottom of the
tubes. The tubes were then incubated for 48 hours at 37ºC.

Casein Hydrolysis:

Plate count agar with sterile skimmed milk was aseptically inoculated using a sterile
inoculating loop full of unknown bacterial culture that was placed in the center of the plate and
spread in a circular fashion. The plate was incubated for 48 hours at 37ºC.

Gelatin Hydrolysis:

Nutrient gelatin deep tube was aseptically inoculated with the unknown bacterial culture by
stabbing the medium ¾ to the bottom using a sterile inoculating loop. The tube was incubated
for 48 hours at 37ºC. After incubation, the tube was removed and placed into a refrigerator for
30 min at 4ºC until the bottom re-solidified and was removed and gently slanted.

IMVIC Tests:

Methyl Red Test:

A methyl red broth was inoculated with 2/3 of the unknown bacterial culture and incubated for
48 hours at 37ºC. A volume of 0.2 mL of methyl red indicator was added to the tube to observe
a colour change of red for a positive result.

Indole Production:

A SIM agar deep tube was aseptically inoculated with the unknown bacterial culture by using
a sterile inoculating loop to stab the medium. The tube was then incubated for 48 hours at 37ºC.
After incubation, 0.5 mL of Kovac’s reagent was added and shaken gently.

Voges-Proskauer Test:

A third of the bacterial culture was aliquot to an empty tube and 0.6 mL of Barritt’s solution.
A and 0.2 mL of solution B was added to the tube. The Barritt’s solution contained 40% KOH
and 5% of α-napthol solution in absolute ethanol. It was then shaken vigorously to allow
aeration. A red colour should appear to indicate a positive test for acetoin.

Citrate Utilization Test:

A Simmons citrate agar slant tube containing sodium citrate, NH4+ and the pH indicator
bromothymol blue was aseptically inoculated using a sterile inoculation loop to perform a stab-
and-streak inoculation technique. The tube was then incubated for 48 hours at 37ºC.

Lysine and Ornithine Decarboxylase Test:

Two tubes labelled as lysine and ornithine containing a medium with the components of the
amino acids lysine and ornithine, glucose and a pH indicator named bromocresol purple was
aseptically inoculated with the unknown bacterial culture using a sterile inoculating loop. Using
a sterile Pasteur pipette, 100 µL of sterile mineral oil was layered on top of the inoculated
media. The cultures were incubated for 48 hours at 37ºC.

Phenylalanine Deamination:

Phenylalanine deaminase slant tube was aseptically inoculated with the unknown bacterial
culture using a sterile inoculating loop by stabbing the agar to the bottom of the medium. The
tube was then incubated for 48 hours at 37ºC in the incubation room. A Pasteur pipette was
used to add 100 µL of 10% aqueous ferric chloride solution on the growth present.

Nitrate Reduction:

A nitrate broth containing 0.5% potassium nitrate was made in two tubes labeled experimental
and control tube. The experimental tube was aseptically inoculated with the bacterial culture
using a sterile inoculating loop and the control was not inoculated. The tubes were then
incubated for 48 hours at 37ºC. After incubation, the presence of growth was observed in the
experimental tube and not in the control tube. 0.5 mL of nitrate test reagents A and B containing
sulfanilic acid and N, N-dimethyl-1-naphthylamine was added.

Catalase Activity:

A tryptic soy agar slant containing the unknown bacterial culture was set in an inclined position
and several drops of 3% hydrogen peroxide were pipetted over the slant.

Urease Activity:

Two tubes containing medium with urea and a pH indicator named phenol red was used to
distinguish members of Proteus from other non-fermenting enteric bacteria. The experimental
tube was aseptically inoculated with the unknown bacterial culture using a sterile inoculating
loop and the control tube was not inoculated. The tubes were then incubated for 48 hours at
37ºC. After incubation, an accumulation of ammonia turns the medium alkaline resulting in an
orange-red to deep pink.

Oxidase Test:

A tryptic soy agar plate medium was aseptically streaked with a single line of unknown
bacterial culture using a sterile inoculating loop. The plate was then incubated in an inverted
position for 24-48 hours at 37ºC. After incubation, 2-3 drops of the oxidase reagent containing
tetramethyl-ρ-phenylenediamine dihydrochloride was added to the surface of the bacterial
growth colonies in the plate. The colour change of the colonies from pink to purple then dark
purple indicates the presence of free oxygen and oxidase in the medium within 20-30 seconds.

API 20E Strip Test:

A 100 µL of unknown bacterial culture was added to each well. The wells with underlined
names had an additional 100 µL added to it and the wells with the names in a box had 100 µL
of mineral oil added. The strip was then incubated for 48 hours at 37ºC.
RESULTS

Table 1: API 20 E Test Results for Unknown Organism

Tests Substrate Reaction Tested Negative Positive Unknown

Results Results organism
Results
ONPG ONPG Beta-galactosidase Colourless Yellow -
ADH Arginine Arginine dihydrolase Yellow Red +
LDC Lysine Lysine decarboxylase Yellow Red +
ODC Ornithine Ornithine decarboxylase Yellow Red +
CIT Citrate Citrate utilisation Yellow/Gre Blue +
en
H2S Na thiosulphate H2S production Colourless/ Black +
Gray Deposit
URE Urea Urea hydrolysis Yellow Red -
TDA Tryptophan Deaminase Yellow Brown-Red -
IND Tryptophan Indole production Yellow Red (2 min) -
VP Na pyruvate Acetoin production Colourless Pink/Red -
(10 min)
GEL Charcoal gelatine Gelatinase No Black Black -
Diffusion Diffusion
GLU Glucose Fermentation/Oxidation Blue Yellow +
MAN Mannitol Fermentation/Oxidation Blue Yellow +
INO Inositol Fermentation/Oxidation Blue Yellow -
SOR Sorbitol Fermentation/Oxidation Blue Yellow +
RHA Rhamnose Fermentation/Oxidation Blue Yellow +
SAC Saccharose Fermentation/Oxidation Blue Yellow -
MEL Melibose Fermentation/Oxidation Blue Yellow +
AMY Amygdalin Fermentation/Oxidation Blue Yellow -
ARA Araninose Fermentation/Oxidation Blue Yellow +

DISCUSSION AND CONCLUSION

The purpose of the experiments performed was to determine the identity of the unknown bacteria
provided with the use of biochemical methods of indication. Four tests namely B-galactosidase test,
the triple iron sugar, a test for starch hydrolysis as well as a plate count agar that contained sterile
milk was performed on the bacteria.

H2S production and motility were performed as well as the gelatine hydrolysis test, lipid hydrolysis
and the IMVIC test. The determination of motility and H2S production is due to the hydrolysis
proteins into amino acids. The bacteria was tested to determine whether it possessed a proteolytic
enzyme gelatinase which breakdown gelatin. The organism tested negative for the enzyme. The
purpose of the test is to determine whether bacteria are able to use gelatine as a nutrient source
and determine whether pathogenically it can breakdown tissue collagen.

IMVIC test is divided into a serious of tests performed to determine the presence of the enzyme
tryptophanase. This enzyme hydrolyses the tryptophan amino acid present in majority of proteins.
Once tryptophan is broken down the Kovac’s reagent is able to identify the product produced which
is indole. In an investigation on new species of enterobacteriaceae, it was cited that it was in
agreement with a positive methyl red test and reaffirmed our error in our API test. However,
everything else in terms of biochemical test matched up perfectly (Farmer et al, 1984).

Our bacteria tested negative for indole production. Next we executive the methyl red test which
basically detects pH changes. We had a negative result and this means that our bacteria are a
Butandiol fementer. Following this experiment we conducted the Voges-Proskauer test which tests
whether or not bacteria can ferment glucose. We received a negative result. Lastly, we conducted
the citrate utilisation test which determines whether or not bacteria can ferment glucose. We
received a negative result. Lastly, we conducted the citrate utilisation test which determines
whether or not a bacteria can utilise citrate as its only source of carbon for its energy purposes and
we have received a negative result yet again for this test. There was one inconsistency involving the
experiments which will be the Voges-Proskauer test. A test for new species that was conducted by
the Journal of Clinical Microbiology matched our results perfectly however yet again there was a
positive result showing that a clear error was made during the API testing on our account (Farmer et
al, 1984).

The organism tested was negative for indole production. Citrate utilisatiion is used to determine
whether bacteria are able to use citrate as source for carbon in order to produce energy . This
showed a positive result.

One article published in the early 80’s stated that certain strains including S. sonnei to name one in
particular were incorrectly identified by Analytab Systems. The test conducted was the test for H2S
(Washington and Martin, 1973). Another article written about the same time showed 100%
accuracy in identifying Shigella with API systems as well as conventional identification methods
(Smith et al, 1972).

We have also for surety a utilised API stripped to test for a wide range of biochemical properties and
enzyme producing indicators. We have found that our bacteria have a positive result for beta-
galactosidase, an enzyme that aids in the breakdown of carbohydrates by fermentation.

S. sonnei is a pathogenic disease and mainly causes diarrhoea, boldly stools and fever as well as
abdominal cramps. In all honesty miniscule hindrances however infection can escalate to severe
neurological symptoms and eventually damage. It causes over 200 million infections in children
under the age of five years with occurrences in adults being exceptionally rare. The only host for the
bacteria is humans and it is seen that on the rare occasion that infection does occur it occurs in gay
men that has sex with other men (Schuster HJ, 2018). Treatment for Shigella usually involves
common antibiotics like ampicillin and amoxicillin the latter being less successful than the first.
However; certain strain of S.sonnei showed resistance to ampicillin and in fact 94% of S.sonnei
tested resistant to the antibiotics, stronger antibiotics where then recommended. However
metabolically this bacteria is not adept to many forms of nutrition uptake (I Rahman, 1994). Lastly it
can be said that this bacteria causes great discomfort among countries healthcare for the millions of
children it affects however it really cannot be seen as a serious threat and is in no way life
threatening.