Nykpingsgymnasium

Natural sciences programme

17/4-13

Acanthamoeba castellanii
show poor symbiosis with
bacterial Spp from estaurine
environments in Nykping

Jhonel Palomino
NV10a
A study on interaction between eukaryotic and prokaryotic cells
Eva Lindblom
Salah Shanan

Abstract
Acanthamoebae castellanii serves as an environmental host for bacterial spp during inconveniences. Amoebae enhance the surviving skills of bacterial spp. Furthermore, the impact of global warming on water temperature leads to bacterial rates and increased infections caused by diverse bacteria. Samples were retrieved from the river of Nykping. The Samples were identified for
A.castellanii by means of DNA extraction, PCR and gel electrophoresis. The microbial identification was performed according to a typing schedule. In addition, co-cultures of A.castellanii and
bacterial spp were examined by an interaction assay which consisted of a cell count. The result
showed that the growth of A.castellanii was suppressed by the presence of the bacterial spp.
The aim of this study was to detect Acanthamoeba castellanii in estuarine environments in Nykping and investigate if they lived symbiotically with bacterial spp.

Table of content
1. Introduction
1.1. Background
1.2. Purpose and aim
1.3. Question formulation
1.4. Limitations
1.5. Factual background
2. Material and Methods
2.1. Reading materials
2.1.1. Sample collection
2.1.2. DNA extraction and PCR
2.1.3. Gel analysis of PCR product
2.2. Isolation and identification of microbals
2.2.1. Gram staining
2.2.2. Biochemical identification of selected isolates
2.2.3. Catalase, DNase and oxidase
2.2.4. Amoeba culture
2.3. Bacterial strains
2.3.1. Culture media, growth conditions and analysis
2.3.2. Microscopy analysis
2.4. Limitations
3. Results
3.1. Gel electrophoresis
3.2. API test
3.3. Amoebaculture
3.4. Catalase, DNase and oxidase
3.5. Growth of A.castellani
4. Discussion
5. Acknowledgements
6. References
7. Appendix
7.1. Appendix I
7.2. Appendix II
7.3. Appendix III
7.4. Appendix IV
7.5. Appendix V
7.6. Appendix VI
7.7. Appendix VII
7.8. Appendix VIII

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1. Introduction
1.1 Background
The summer of 2012 I had the opportunity to attend Karolinska institute Summer Research School
together with 20 other students from different parts of Sweden. I was handed two supervisors, Amir
Saeed and Soni Priya Valeru whom I worked with. Amir and Soni work at the department of laboratory medicine in Gunnar Sandstrm group where they investigate the interaction between amoeba and
diverse bacteria. They have discovered that bacteria can live intracellularly in amoeba, evading its
phagocytosis. I was involved in a project where I was going to examine the role of the Outer Membrane protein A in Vibrio cholerae in interaction with Acanthamoeba castellanii.
Since the amoebae serves as an environmental host for bacteria it can work as a pathogen for humans
causing diverse infectious diseases.
I found this study interesting and wanted to continue studying the interaction between eukaryotic and
prokaryotic cells. That was how I came up with the idea of doing a similar study on the river of Nykping (Nykpingsn) and adjacent waters.
But, not only amoeba enhances the growth of bacteria. The climate change i.e global warming affects
the growth of bacterial species leading to a higher concentration of bacteria in aquatic environments.
Increased water temperature and interaction with amoeba may enhance the growth of bacteria significantly. That means that bacterial growth in aquatic environments is benefitted both from global warming and presence of amoebae. Moreover, recent studies show that V. cholerae ratings in the Baltic Sea
have increased.

1.2 Purpose and aim

The aim of this study is to determine if Acanthamoeba castellanii is present in the waters adjacent to
Nykping and if A. castellanii is able to live in symbiosis with the diverse bacteria.
This research will disclose if amoebae are able to serve as an environmental host for bacterial species
in waters of Nykping, it is known that Acanthamoeba enhances the living skills of some bacteria, e.g.
Vibrio, Pseudomonas, Shigella, Legionella and Salmonella spp. Thus, does not only the amoeba enhance the growth of the bacterial species, nevertheless, global warming is also represented as a factor.
Moreover, amoeba containing bacteria can cause infections. Such infections are already known e.g.
meningitis and encephalitis.

1.3 Question formulation

In order to determine if Acanthamoeba castellanii and bacteria live in symbiotic relation in the river of
Nykping one will have to answer these questions:
Is Acanthamoeba castellanii present in the river of Nykping?
Which species of bacteria are the most common in the river of Nykping?
Can the particular bacteria interact with A. castellanii (in vitro)?
Do Amoeba and bacteria live in symbiotic relation in estuarine environments in Nykping?

1.4 Limitations
In this study I will not examine how the biotic or the abiotic factors affect the presence of both amoeba
and bacteria. Biotic factors such as predators, excess of smaller organisms that serve as food or other
organisms will not be considered in this study. Furthermore, abiotic factors such as light, water temperature, salinity, turbidity, pollution or the lack of certain elements e.g. sulfur or nitrogen will neither
be considered in this study. The oxygen level is presumed to be aerobic.

Moreover, this study was performed with water samples from the Baltic Sea but only with water
sources from Nykping. The amount of water sample is limited to 1.2 L from each source.

1.5 Factual background

Acanthamoeba species have been found in a large variety of sources, amongst them: lakes, swimming
pools, tap water, bottled water, dialysis machines, contact lens equipment, soil, fresh, brackish and sea
water (8-10). Normally, amoeba feed on different bacteria by phagocytosis. Still, some bacterial species are capable of surviving and multiplying inside amoebae(7,10-12). Furthermore, amoebae containing bacteria can cause infections and these infections occur worldwide (7-12). Acanthamoeba castellanii has two life stages, a trophozoite and cyst stage (7-10). The amoeba normally eats and divides
in the trophozoite stage. Whereas the cyst stage is a dormant phase in which bacteria can linger, often
during environmental inconveniences (7,9-13). The cyst also serves as a transporter for bacteria from
one host to another (7-10). Furthermore, the amoeba in cyst form can gain entry to the body through
various means and while in cyst form it can be airborne and viable for several years (7-9).
Cronobacter species (formerly known as Enterobacter sakazakii) are Gram-negative, rod shaped, motile bacteria of the Enterobacteriaceae family(nonspore-forming)(1-5). Cronobacter species consist of
seven genomospecies; Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii,
Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti(1,4,5). Bacteria belonging to the Cronobacter genus are commonly found in waters, various
foods, soil, plant material and PIF(Powdered Infant Formula)(1-3,6). PIF is a substitute for breast milk
feeding and its matrix supports the growth of Cronobacter spp(3-6). In addition, Contaminated PIF has
been linked with infections in neonates, primarily causing meningitis(1-5). However, Cronobacter
species can also cause other infections in infants such as septicemia, necrotizing enterocolitis, NEC,
with the mortality rate in some cases being 80%(1-5). Infections in immunocompromised adults has
also been noted(2-4). Furthermore, Cronobacter species are more thermotolerant than other members
of the Enterobacteriaceae. They are able to grow at a range of 6-67C with the optimal range being 3743C.(2-4). Numerous studies disclose the OmpA as a contributory virulence factor. OmpA in Cronobacter species aids the infection by facilitating the penetration of the blood barrier, furthermore, inducing microtubule condensation(2-4). A study suggests that OmpA expressing(Cronobacter) are able to
cross the intestinal barrier and bind to brain epithelial cells(2). Whereas Cronobacter isolates with a
lack of expression of OmpA could not bind to epithelial cells(2,3). Additionally, Cronobacter spp utilizes biofilm formation which facilitates its survival and it produces an enterotoxin, but, the importance of the toxin is still unclear(3-5).
Bacillus subtilis is a Gram-positive, rod-shaped spore forming bacterium that is commonly found in
soil and plant material(16,18-19). Some studies suggests that Bacillus subtilis dwells in both the gut
and soil(19,21). In addition, B. subtilis is considered as a non-pathogenic bacterium, nevertheless, it is
capable of causing gastrointestinal disease.(21). The bacterium supports plant growth and is used as a
biocontrol agent, it also comprises a higher activity of amylase(16,20,22). B. subtilis is capable of
forming spores and biofilms. It has been used in several studies in order to determine certain molecular mechanisms(16,18). A biofilm is when bacteria are encased in a self-produced extracellular matrix.
The biofilm protects the bacteria. Furthermore, Bacillus subtilis is able to form spores. The endospore
protects the genome during harsh conditions and heat stress(17,21). While in spore form the bacteria
are able to survive for a long period of time in harsh and nutrient-free environments. The protective
case is composed of a pigment that is similar to melanin, it protects the cell from UV radiation and it
has been shown that melanin can interfere with phagocytosis(17). Bacillus subtilis is frequently used
for industrial applications as a protein secreting microorganisms(18)
Climate change affects temperature rates in the Baltic sea. The increase in temperature leads to higher
wound infections caused by Vibrio spp(14,15). A study made in the Baltic sea showed that the average
increase in temperature per decade is 1.0C which is estimated to be seven times higher than the global
average. In addition, Vibrio associated infections, as well as the SST(Sea Surface Temperature)
peaked during the hot summers of 1994, 2003 and 2006(14). The increase in infections is partly due to
the more extensive replication of bacteria, nevertheless, leisure activities are increased with the in5

crease in temperature(14,15). Two of the location sites where the water samples were retrieved from
are well-visited for bathing during summer.
The aim with this study is to disclose if A.castellanii can live in symbiotic relation with bacterial species from estuarine environments in Nykping.

2. Material and Methods

2.1 Reading material
Reading material was retrieved from PubMed database(full access version), Nature reviews and microbiology books.

2.1.2 Sample collection

Four water samples of 1.2 L each was collected from the river of Nykping (Nykpingsn). The collection sites were: Piren, Hllet, ngstugan and Tjuvholmen. Map, Appendix I.
The content from each sample was centrifuged in 50 ml tubes at 4000 rpm for 5 minutes. In order to
obtain the microorganisms the pellet was transferred to a clean 50 ml tube and the overflow was discarded. The same procedure was performed with all water samples and the pellet from the same collection site was put to the same 50 ml tube.

2.1.3 DNA extraction and PCR

50 ml of water from all 50 ml tubes(samples) was centrifuged for 10 min at 4000 rpm. The pellets
were used for DNA extraction using Qiagen DNA mini kit (Qiagen, Valencia, CA, USA). All samples
were amplified with PCR. The same procedure was performed with all samples. For detailed methods,
see Appendix II.

2.1.4 Gel analysis of PCR product

PCR conditions were: 30 cycles of 94C (denaturation) for 1 min, 50C (annealing) for 1 min, 72C for 1
min (extension) and 72C for 10 min (final extension). Following, the PCR products were analyzed by
gel electrophoresis with agarose gel in 1x TBE buffer (Tri base, boric acid and EDTA (pH 8.0)). The
gel was later stained in 0.1% SYBR Green bath in order to visualize it in UV translumination. The gel
was photographed using Polaroid films. For detailed methods, see Appendix II and III
2.2 Isolation and identification of microbials
A typing schedule was used as guidance to identify the bacteria. See Appendix IV.
All 50 ml tubes containing water were vortexed, subsequently, 10 L from each sample was streaked
on both CLED and blood agar plates and incubated aerobically at 37C for 18 hours. Later, selected
isolates from all blood agar plates were transferred to new blood agar plates and incubated at 37C for
18 hours. Three isolates were selected from each Hllet and Piren, and five isolates were selected from
each Tjuvholmen and ngstugan.
2.2.1 Gram staining
To determine the morphology and whether the isolates were Gram positive or Gram negative a gram
staining was performed. All isolates were investigated under a light microscope. For a detailed method, see Appendix V.
2.2.2 Biochemical identification of selected isolates
Four presumptive Enterobacteriaceae isolates were investigated with the API 20E test
kit(BioMrieux, France). The result was acquired from the API 20E database. For a detailed method,
see Appendix VI.

2.2.3 Catalase, DNase and oxidase

A presumptive Staphylococcus aureus isolate was investigated with a catalase and DNase test. Two
colonies(Tjuvholmen 5) were transferred to 5 ml 3% H2O2. Additionally, the presumptive isolate was
streaked on a DNase agar plate together with a positive control. Add two-three colonies from isolate(ngstugan 4) to a filter paper and add oxidase enzyme.

2.2.4 Amoeba culture

All tubes containing water samples were vortexed and 1 ml (water) from each tube was added together
with 500 l gentamycin (250g/ml), Streptomycin (50g/ml) and antifungal to 50 ml cell culture
flasks. All samples were incubated in room temperature for 2 hours. 10 ml ATCC medium no. 712
was added and shaken, finally, incubated at 30 C. All samples were continuously viewed under microscope.

2.3 Bacterial strains

Bacterial strains used in the interaction study are Enterobacter sakazakii(Cronobacter Spp) and Bacillus subtilis.

2.3.1 Culture media, growth conditions and analysis

All bacterial strains were grown at 37C for 16-18 hours. A. castellanii was grown in ATCC medium
no. 712 at 30C to a concentration of 105 cells ml-1. The bacterial strains were grown at 37C in LB
broth to an absorbance of 0.6 at 600nm. Co-cultures of each bacterial strain and A. castellanii were
incubated in six well plates (Corning Incorporated Costar) filled with 3 ml of ATCC medium no. 712
containing an initial concentration of 105 cells ml-1 of A. castellanii and 106 cells ml-1 of each bacterial
strain. For a detailed method, see Appendix VI.

2.3.2 Microscopy analysis

Acanthamoeba castellanii cells in the absence and presence of bacteria were stained with Erythrosine
B staining and counted in a Brker chamber under a light microscope. For a detailed method, see Appendix VII.

2.4 Limitations in material and methods

Only three colonies from Piren and Hllet and five colonies from Tjuvholmen and ngstugan were
selected for the microbial identification. Additional tests i.e API 20E, catalase, DNAse and oxidase
was performed on presumptive isolates according to the typing schedule. Isolates Piren 2, ngstugan
1, Tjuvholmen 2 and Hllet 2 were selected for the API test. Isolate ngstugan 4 was selected for an
oxidase test. Finally, was isolate Tjuvholmen 5 selected for further analysis with catalase and DNase.
All isolates were grown on blood agar medium since it is a rich medium and it supports the growth of
diverse bacteria. CLED was used to determine lactose fermentation.

3. Results
3.1 Gel electrophoresis
The result showed that all samples contained Acanthamoeba castellanii. The sample from Piren
showed less concentration of DNA but a small/light
stripe could be observed(fig1).

3.2 API test

The Analytical Profile Index 20E identified the isolates as Enterobacter sakazakii (Cronobacter
cies) with 98.4% coincidence, and as Vibrio
7

Fig 1. Gel analysis of collected water samples from Nykping,

DNA fragment 487bp. Ladder is marked with L. PCR product
from ngstugan is marked with . PCR product from Piren is
marked with P. PCR product of Hllet is marked with H. PCR
product of Tjuvholmen is marked with T. Positive control is
marked with C.

lyticus with 72% accuracy. Remaining isolates

could not be determined
by the API 20E test.

3.3 Amoeba culture

Fig 2. Growth of Acanthamoeba castellanii. Number of A.castellanii in absence of B.subtilis and

Cronobacter Spp (green line). Number of A.castellanii in presence of Cronobacter Spp (red line).
Number of A.castellanii in presence of B.subtilis (blue line).

The amoebae grew badly,

however, three out of four
samples showed growth
the following week. The
sample from Piren did not
grow. All amoeba had
died one month after the
amoeba culture had been
performed.

3.4 Catalase, DNase and oxidase

The catalase test was positive and the DNase test was negative. The oxidase test was positive.

3.5 Growth of A. castellanii

The growth of Acanthamoeba castellanii in presence and absence of Bacillus subtilis and Cronobacter
Spp was studied by cell counts in a Brker chamber. The amoebae were stained with Erythrosine stain
and dead amoeba obtained a red color complex. In addition, it was found that co-cultured amoebae
died after 5 days of incubation, whereas amoeba in absence of bacteria kept growing after seven days
of incubation(fig 2).
Table 1, Microbial identification based on Colonial morphology, Gram stain, Morphology and Additional tests.
*Estimated species, according to type schedule for aerobic and facultative anaerobic bacteria.
Source/isolate

Colonial morphology

Gram
Stain

Morphology

Species*

Piren 1
Small white
GRod+long chain
Piren 2
Large white
GRod
Enterobacter sakazakii(98.4%)
Piren 3
Large yellow
G+
Rod+diplococcus
Micrococcus Spp
ngstugan 1
Small white
GRod
Negative test
ngstugan 2
Large pale(hemo)
G+
Rod
Bacillus subtilis
ngstugan 3
Large yellow
G+
Diplococcus
Micrococcus Spp
ngstugan 4
Large white
GRod(small)
Pseudomonadaceae
ngstugan 5
Large pale
G+
Diplococcus
Micrococcus Spp
Tjuvholmen 1
Small white
GRod
Tjuvholmen 2
Medium pale(hemo)
GRod+long chain
Vibrio alginolyticus (72.4%)
Tjuvholmen 3
Large white
G+
Rod
Bacillus subtilis
Tjuvholmen 4
Large yellow
G+
Diplococcus
Micrococcus Spp
Tjuvholmen 5
Medium white
G+
Coccus
Staphylococcus epidermidis
Hllet 1
Small white
GRod+long chain
Hllet 2
Medium white
GRod
Negative test
Hllet 3
Large yellow
G+
Rod
Bacillus subtilis
*Estimated species, according to type schedule for aerobic and facultative anaerobic bacteria.

Additional test

API
API

Oxidase

API

Catalase, DNase
API

Discussion
The information which was used in this study is acquired from scientific articles, mainly from PubMed. PubMed is a database with both recent and old articles on research in medical and biological science. The information acquired is verified by reading different articles supporting the same information.
The methods used for the identification of A.castellanii are conventional, but, well valued. The PCR
was performed in order to amplify the possible amount of amoebic DNA. The PCR is of importance
since there was no knowledge about the presence or absence of amoeba. The gel electrophoresis
showed positive results meaning that the method 2.1 was performed well. If the opposite result would
be seen, then it may be suggested that either the DNA extraction or the PCR was not properly performed. The gel electrophoresis is a good test since it is not very likely that it may become contaminated, and, if properly performed one obtains good results.
The typing schedule which was used for the bacterial identification was acquired from Mamun Ur Rashid. The schedule limits the bacterial identification to facultative anaerobic and aerobic bacteria. The
typing schedule considers the probability of the bacteria, but, in order to be certain one needs to complement with additional tests. Additional tests can be biochemical identification and enzymatic activity
tests. A correction on the typing schedule is that oxidase-positive is supposed to be Pseudomonadaceae not Pseudomonas aeruginosa.
All samples were vortexed and grown on both blood and CLED agar plates. Blood agar is a rich medium which supports the growth of diverse bacteria and differentiate the hemolytic bacteria. CLED agar
is used in order to differentiate the bacterial ability to ferment lactose. It was determined to grow on
both CLED and blood agar medium since the composition of CLED does not support the growth of
certain bacteria.
The gram staining was performed in order to determine if the bacteria were gram positive or negative.
The morphology was observed under immersion objective. The gram staining should have been performed twice in order to confirm the stain. If the gram staining is not properly performed it may be
decolorized, gram positive bacteria will look like gram negative and gram negative will appear pink.
The API test is a biochemical test which includes 20 enzymatic activity tests combined in a strip. It
consists of 20 tubes with different content and after having added the bacterial suspension one was to
observe the reactions. Different bacteria are able to produce different enzymes and the reactions on the
strip show if the bacteria possess that particular enzyme. According to the positive and negative results
one can determine at the species level, non fastidious gram-negative rods by comparing the test result
numbers to a database. The bacteria chosen for the API test where assumed to be gram-negative and
oxidase-negative.
The oxidase test was only performed on the sample from ngstugan 4 since we were not sure whether
the isolation was an Escherichia coli or not. Mamun suspected the bacteria to be Escherichia coli
since it obtained the right properties, however, the result showed the opposite. Furthermore, additional
tests were needed in order to confirm it as Pseudomonas aeroginusa, which it was suggested as according to the typing schedule. The material needed in order to perform the further analysis was not
available.
The aim of this study was to determine whether amoebae and bacteria could live in symbiosis in
Nykping, an attempt to grow amoebae from my samples was made. The plan was to perform the interaction with amoebae collected from Nykping, unfortunately we did not manage to grow strong
amoebae i.e amoebae that remained viable in ATCC medium. We later used bought A. castellanii for
the interaction.

The bacterial spp used in the study were Enterobacter sakazakii and Bacillus subtilis. These were used
due to the poor growth of the other bacteria. This study would have been more interesting if V. alginolyticus, Staphylococcus epidermidis or the Pseudomonadaceae would have been co-cultured with
amoebae, since they are pathogenic. The poor growth of both V. alginolyticus, S. epidermidis led to
the obligate use of Bacillus spp which was the only one together with E. sakazakii that grew. The poor
growth can be explained by the storage of the isolates. When not used, the isolates were stored in the
refrigerator, when we later tried to subculture them again they did not grow. The cause of the poor
growth may have been that I stored them in 5C instead of -20 for a long time.
The reliability on the method used for the identification of bacteria should be discussed since this was
the most intricate part of this study. There are a vast variety of methods in order to determine bacteria
at the species level. Firstly, one needs to subculture the bacterium twice or trice in order to obtain pure
strains. Secondly, one performs the gram stain in order to determine whether the bacterium is gram
positive or negative and to determine the morphology. When a pure strain is obtained and one has determined the gram stain and the morphology one can proceed with a whole genome sequence, a
MALDI examination or additional tests. The whole genome sequence is based on the identification of
the order of the four nucleotides within a DNA molecule. After having determined the order one can
compare it to already known combinations and determine the species of the bacteria. The MALDI examination is a type of mass spectrometry, where the bacterium is determined by ionizing of a particular compound. The bacterial identification may also be performed as I have done it, according to a typing schedule, with additional biochemical tests as the API or ID. If one wants to obtain an accurate
result is the MALDI and the whole genome sequence preferred. A combination of these methods also
gives an accurate result. These are suggested methods that one can use if one is ought to determine the
species of a bacterium from a sample. However, if one is searching for a particular bacterium, then one
can apply another methodology. If one is searching for a particular bacterium e.g Staphylococcus aureus then one can grow isolates from the sample in differential and selective medium. The differential
and selective medium distinguishes the bacterium by expressing certain properties such as catalytic
enzymes. After having grown the isolate on several differential and selective medium one can confirm
the result by performing a biochemical test. This methodology is mainly used in research when looking for a particular bacterium.
In my project plan I included a viable count assay and an intracellular assay, these were not included
in this study. In my project plan I included the viable count assay and intracellular assay in order to
perform a proper study on the interaction.
I planned to use additional methods to examine how much the amoeba affected the growth of bacteria
in relation to how much the growth of bacteria affected the growth of amoeba. One can determine
whether the growth of bacteria inhibits the growth of amoeba or whether the growth of bacteria does
enhance the growth of amoeba by means of viable count assay. The viable count is used in order to
estimate the growth of bacteria by culturing them on blood agar and determine the concentration of
bacteria by counting the observed colonies. The only way to determine the bacterial growth in my
study was by observation of the turbidity in the samples. The turbidity is mostly made of the increase
of bacteria, but also due to decay products of the dead amoebae and bacteria.
The intracellular assay can be used in order to determine whether the bacteria have infected the amoebae or not. This assay includes isolation of amoeba, gentamycin(antibiotic) treatment, growth of extracellular and intracellular bacteria and count of observed colonies.
The extensive methodology of this project should be considered while performing it. The time planning was well evaluated, however, did my supervisor not follow it, leading to a post-poning story with
no end. This study should preferably be performed daily instead of once a week or twice a month. It
would be easier since one would have fresh strains, no pressure in regards of extra studying and a continuous way of working. The budget should neither be restricted as in my case in terms of the additional planned assays.

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The gel analysis showed that three out of four samples were strongly positive. This suggests
the presence of A.castellanii in estuarine environments in Nykping. The sample from Piren did not
show a strong result, nevertheless, there is a small stripe that can be observed. This small stripe may
imply that the presence of A.castellanii is not as vast in comparison to the other sources. Another possible reason for the small stripe may be, wrong while performing the DNA extraction, PCR, or while
applying the sample to the gel, as explained above. In addition, the presence of amoebae may suggest
A.castellanii as an environmental host for some bacterial spp in Nykping.
The API test showed two out of four identifiable results. The isolates from ngstugan 1 and Hllet 2
showed negative results, the bacterial strains were unable to perform any reaction on the strip. However, the isolates from Piren 2 and Tjuvholmen 2 showed positive results and were identified as Enterobacter sakazakii and Vibrio alginolyticus. The reason for the negative/unidentifiable results may be,
that the bacteria were not properly stained or that the bacteria were not oxidase-negative. This is the
main reason for why I should have performed the Gram stain twice, as suggested above. If the bacteria
were not properly stained or oxidase-positive, the API 20 test kit is not an option for determination of
the species. The results should be confirmed by performing more tests in order to be accurate that the
isolates are as implied by the API test. The confirmatory tests can be implemented as previously explained.
In addition, the high presence of V.alginolyticus is interesting. Two of the articles that I have read suggests an emerging increase in Vibrios, leading to an increase in infections caused by Vibrios. The presence of V.alginolyticus in lower temperate waters is further confirmed by the study made by Schets et
al(15). The possible presence of V.alginolyticus in Nykping is of importance since it may further verify the increase of Vibrios as implied by the studies made in the Baltic sea and the Netherlands(14,15).
The amoeba culture from my samples showed poor growth and they eventually died. One sample did
not show any growth at all, the sample collected from Piren. This may explain the small stripe on the
gel electrophoresis, there may not have been any amoeba in Piren. The lack of amoebae in Piren may
be due to the restricted amount of water collected. I only collected 1.2 L, maybe there were no amoebae in that amount, one cannot be certain that Piren is lacking amoebae. All amoebae were incubated
in ATCC medium, however, they did not grow, implying that A.castellanii is unable to show symbiotic effect. A prediction can be that the amoebae may not be strong or satisfied with the nutrient, further investigation on why the amoebae did not survive is needed.
The catalase and DNase tests were performed on the isolate from Tjuvholmen 5 according to the typing schedule. The catalase was positive and the DNase was negative, this means that the isolate from
Tjuvholmen 5 is Staphylococcus epidermidis. The oxidase test was performed on the isolate from
ngstugan 4 and it was determined to Pseudomonadaceae, a correction on the typing schedule. These
bacteria were planned to be used in the co-culturing, but due to the poor growth they were excluded
from the study, as described above.
The cell count showed that bacteria suppressed the growth of amoebae after two days of incubation.
The amoebae in absence of bacteria grew to an extent of 6.5 log of cfu. The slight decrease of
A.castellanii after one day of incubation may be due to an improper mixing. Amoebae in absence of
bacteria should not decrease since they have no predators or any competence at all in the well plate.
The reason for the slight decrease may be the improper mixing. Before starting the cell count, one
needs to mix the ATCC and amoeba thoroughly in order to obtain the amoebae that are stuck at the
bottom and walls.
The discovery of the decrease of amoebae while co-cultured with Cronobacter spp and B. subtilis is of
interest since it shows the opposite of several studies. It has been shown that A.castellanii serves as an
environmental host for diverse bacterial spp, e.g Pseudomonas aeroginusa, Vibrio cholerae, Vibrio
mimicus, Franciscella tularensis, Legionella pneumophila, Mycobacterium avium, Shigella sonnei etc
(7,10-13). However, it has been stated that strains of Pseudomonas aeroginusa are able to kill and inhibit the growth of A.castellanii with exoenzymes(13). This means that the extensive production of
11

enzymes and toxins by P. aeroginosa can kill amoebae. The enterotoxin produced by Cronobacter spp
may kill amoebae. If one can state the increase of bacteria in the well plate, then one may assume that
the concentration of the enterotoxin is further increased. Enterotoxins have cytotoxic effects, this may
explain why the amoebae died after five days of incubation. The reason for the decrease of amoebae
while co-cultivated with B.subtilis may be B. subtilis vast survival abilities. Bacillus subtilis are able to
form endospores and biofilms. The spore-formation and biofilm-formation are survival strategies used
by B. subtilis. There is a small chance that there may have been amounts of toxic decay products of
either A.castellanii or B. subtilis. The toxic decay products may kill or inhibit the growth of amoeba. It
is not very likely that this is the cause for the decrease of amoebae, however, it may be a possible reason. Further investigation is needed in order to determine the cause of the decrease of amoebae after
five days of incubation.
The interaction between amoebae and bacteria could have been studied with a more accurate method,
as explained above. One cannot state a trustworthy correlation on the effect of bacteria on amoebae. In
addition, one can conclude that there is poor symbiotic effect between the bacterial spp retrieved from
the river of Nykping and A.castellanii.
Table one show the results from the gram stain, colonial morphology, morphology, additional tests
and the estimated species. A proper spore test was not performed on the bacteria estimated as Bacillus
subtilis, nevertheless, a spore formation was observed in the microscope. The result shows that the
subculture could have been performed twice since all isolates were not pure. Some isolates contained
rod shaped bacteria together with long chain, coccobacilli. Furthermore, the bacterial species were estimated by Mamun Ur Rashid who is a bacteriologist. The estimated species need to be further confirmed with additional tests for an accurate result, as explained above.
One can conclude that the most abundant bacteria are Micrococcus spp and Bacillus subtilis. This is
not particularly surprising since they both are common in soil and water.
The findings from this experiment show that A.castellanii does not live in symbiosis with Cronobacter
sakazakii nor Bacillus subtilis. In order to perform this study properly one needs to perform additional
tests that will confirm the bacterial identification. A more extensive methodology for the interaction
assay is needed in order to conclude how the bacteria affected the amoebae. Lastly, one needs to perform this study without a strongly restricted budget that limits the methods that can be applied.

Acknowledgements
This project was supported by Gunnar Sandstrm group, Karolinska Institute, Stockholm. I wish to
express my sincere gratitude towards my supervisors Salah Shanan and Eva Lindblom who have
helped with my study and validation of ideas. A special thanks to Amir Saeed who has helped when
my supervisor has been absent. Furthermore, I wish to express a special regard towards Mamun ur
Rashid who has helped me with the microbial identification. A special thanks towards Nykping county for letting me analyze the water and providing me with travelling costs.

12

References
1
Cetinkaya E, Joseph S, Ayhan K, Forsythe SJ. Comparison of methods for the microbiological
identification and profiling of Cronobacter species from ingredients used in the preparation of infant
formula.. Mol Cell Probes. 2013 Feb;27(1):60-4. Doi: 10.1016/j.mcp.2012.10.003. Epub 2012 Oct 23.
2
Healy B, Cooney S, OBrien S, Iversen C, Whyte P, Nally J, Callanan JJ, Fanning S Cronobacter (Enterobacter sakazakii): an opportunistic foodborne pathogen.. Foodborne Pathog Dis. 2010
Apr;7(4):339-50. Doi: 10.1089/fpd.2009.0379. Review.
3
Chenu JW, Cox. Cronobacter (Enterobacter sakazakii): current status and future prospects.
JM.Lett Appl Microbiol. 2009 Aug;49(2):153-9. Doi: 10.1111/j.1472-765X.2009.02651.x. Epub 2009
May 27. Review.
4
Yan QQ, Condell O, Power K, Butler F, Tall BD, Fanning S. Cronobacter species (formerly
known as Enterobacter sakazakii) in powdered infant formula: a review of our current understanding
of the biology of this bacterium.. J Appl Microbiol. 2012 Jul;113(1):1-15. doi: 10.1111/j.13652672.2012.05281.x. Epub 2012 Apr 11. Review.
5
Joseph S, Forsythe SJ. Insights into the Emergent Bacterial Pathogen Cronobacter spp., Generated by Multilocus Sequence Typing and Analysis. Front Microbiol. 2012;3:397. doi:
10.3389/fmicb.2012.00397. Epub 2012 Nov 22.
6
Turck D. Safety aspects in preparation and handling of infant food.. Ann Nutr Metab.
2012;60(3):211-4. doi: 10.1159/000338215. Epub 2012 Jun 6.
7
Sandstrm G, Saeed A, Abd H. Acanthamoeba-bacteria: a model to study host interaction with
human pathogens. Curr Drug Targets. 2011 Jun;12(7):936-41. Review.
8

Leo Shapiro. Acanthamoeba Eukarya; Misc CDC parasites: Acanthamoeba castellanii

9
Shanan S, Abd H, Hedenstrm I, Saeed A, Sandstrm G. Detection of Vibrio cholerae and
Acanthamoeba species from same natural water samples collected from different cholera endemic areas in Sudan. BMC Res Notes. 2011 Apr 7;4:109. doi: 10.1186/1756-0500-4-109.
10
Abd H, Valeru SP, Sami SM, Saeed A, Raychaudhuri S, Sandstrm G. Interaction between
Vibrio mimicus and Acanthamoeba castellanii. Environ Microbiol Rep. 2010 Feb;2(1):166-171.
11
Abd H, Saeed A, Weintraub A, Nair GB, Sandstrm G. Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living
amoeba Acanthamoeba castellanii.. FEMS Microbiol Ecol. 2007 Apr;60(1):33-9.
12
Abd H, Saeed A, Weintraub A, Nair GB, Sandstrm G. Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living
amoeba Acanthamoeba castellanii. FEMS Microbiol Ecol. 2007 Apr;60(1):33-9.
13
Abd H, Wretlind B, Saeed A, Idsund E, Hultenby K, Sandstrm G. Pseudomonas aeruginosa
utilises its type III secretion system to kill the free-living amoeba Acanthamoeba castellanii.. J Eukaryot Microbiol. 2008 May-Jun;55(3):235-43. doi: 10.1111/j.1550-7408.2008.00311.x.
14
Baker-Austin, Craig, Trinanes, Joaquin A. Taylor, Nick G. H. Hartnell, Rachel, Siitonen, Anja, Martinez-Urtaza, Jaime. Emerging Vibrio risk at high latitudes in response to ocean warming. Nature Clim. Change. 2013/01//print. 317377. Nature Publishing Group. 1758-678X.
http://dx.doi.org/10.1038/nclimate1628. 10.1038/nclimate1628 9/4-13
13

15
Schets FM, van den Berg HH, Marchese A, Garbom S, de Roda Husman AM. Potentially human pathogenic Vibrios in marine and fresh bathing waters related to environmental conditions and
disease outcome.. Int J Hyg Environ Health. 2011 Sep;214(5):399-406. doi:
10.1016/j.ijheh.2011.05.003. Epub 2011 Jun 12.
16
Vlamakis H, Chai Y, Beauregard P, Losick R, Kolter R. Sticking together: building a biofilm
the Bacillus subtilis way. Nat Rev Microbiol. 2013 Mar;11(3):157-68. doi: 10.1038/nrmicro2960.
Epub 2013 Jan 28.
17
McKenney PT, Driks A, Eichenberger P. The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nat Rev Microbiol. 2013 Jan;11(1):33-44. doi: 10.1038/nrmicro2921.
Epub 2012 Dec 3. Review.
18
van Dijl JM, Hecker M. Bacillus subtilis: from soil bacterium to super-secreting cell factory.
Microb Cell Fact. 2013 Jan 14;12:3. doi: 10.1186/1475-2859-12-3.
19
Schyns G, Serra CR, Lapointe T, Pereira-Leal JB, Potot S, Fickers P, Perkins JB, Wyss M,
Henriques AO. Genome of a Gut Strain of Bacillus subtilis. Genome Announc 2013 Jan;1(1). doi:pii:
e00184-12. 10.1128/genomeA.00184-12. Epub 2013 Feb 14.
20
Nakamura, L. K. Deoxyribonucleic acid relatedness of lactose-positive Bacillus subtilis strains
and Bacillus amyloliquefaciens. International journal of systematic bacteriology, 1987, 37.4: 444-445.
21
Roberts, Diane. Greenwood, Melody. (2003) Practical food microbiology. Malden, Massachusetts USA: Blackwell publishing.
22

Eldor,Paul A. (2007) Soil microbiology, Burlington, Massachusetts USA: Elsevier

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Appendix I
Map of Nykping
Map over the sample collection sites. 1.2 L of samples was obtained from each Hllet, Piren, ngstugan and Tjuvholmen. Sample location sites are displayed with a star.

15

Appendix II
DNA Extraction with QIAamp DNA Mini Kit

Centrifuge 50 ml water for 10 min at 4000 rpm and use the pellets for DNA extraction using
Qiagen DNA mini kit. Place the pellet in a 1.5 ml microcentrifuge tube.
Add 180l buffer ATL and 20l Proteinase K and vortex.
Place the tube in 56C water bath for 2 hours.
Remove from water bath, add 200l buffer AL and vortex for 15s.
Place the tube in 70C water bath for 10 minutes.
Add 200l 100% ethanol and transfer entire volume onto spin column.
Centrifuge at 8000 rpm for 1 minute; discard flow-through. Place the QIAamp Mini spin column in a clean 2 ml collection tube and discard the tube containing the filtrate.
Add 500 l buffer AW1 and centrifuge at 8000 rpm for 1 minute; discard flow-through.
Add 500l buffer AW2 and centrifuge at 14000 rpm for 3 minutes; discard flow-through.

PCR using Qiagen HotStarTaq Master Mix

Reaction setup using HotStarTaq Master Mix
Component
Volume
HotStarTaq Master Mix
125l
Primer 1
1.5l
Primer 2
1.5l
Rnase-free water
97l
Template DNA
25l

Pipette all the components to a small tube, excluding the template DNA. Subsequently, transfer 2 l
template DNA together with 18 l of the Master Mix(HotStarTaq Master Mix, Primers 1 and 2,
Rnase-free water) to a PCR tube. Mix the content gently. This is performed with all four DNA templates.
Place the PCR tubes in the thermal cycler and start the cycling programe.
Cycling conditions
Step
Initial heat activation
Denaturation
Annealing
Extension
Number of cycles(20)
Final extension

Time
15 min
1 min
1 min
1 min
30
10 min

Temperature
95C
94C
50C
72C
72C

16

Appendix III
Agarose gel electrophoresis
Preparing the agarose gel
Measure 2g Agarose powder and add it to a 500 ml flask.
Add 100 ml 1xTAE Buffer to the flask.
Melt the agarose in a microwave until the solution becomes clear.
Let the solution cool to about 50-55C.
Seal the ends of the casting tray with two layers of tape.
Place the combs in the gel casting tray.
Pour the melted agarose solution into the casting tray and let cool until it is solid.
Carefully pull out the combs and remove the tape.
Place the gel in the electrophoresis chamber.
Add enough 1xTAE buffer.
Loading the gel
Add 2 l of 6x Sample Loading Buffer(stain) to each 10 l PCR reaction.
Pipette 10 l of each sample/ sample loading buffer mixture into separate wells in the gel.
Pipette 3 l of the DNA ladder standard into one well(left well).
Pipette 10 l of control into separate well in the gel.
Running the gel
Place the lid on the gel box, connect the electrodes.
Connect the electrode wires to the power supply.
Turn on the power supply to about 120 volts for 30 minutes.
Turn of the power.
Disconnect the wires from the power supply.
Remove the lid of the electrophoresis chamber.
Using gloves, carefully remove the tray and gel.
Gel Staining
Remove the gel from the casting tray and place into the staining dish containing Ethidium
Bromide.
Allow gel to stain for 35 minutes.
Rinse the gel with water to remove residual stain.
View the gel against a transilluminator (An ultraviolet lightbox), which is used to visualize
ethidium bromide-stained DNA in gels.

17

18

Appendix V
Gram staining
Add 100 l of water(sterile H2O) to a slide. Transfer a colony and emulsify it ovally with the added
water. Let the smear dry in air and fix it by sweeping it two times/twice over a flame.
Soak the slide with crystal violet solution and let stand for one minute. Wash it under running
tap water.
Soak the slide with iodine solution and let stand for one minute. Wash it under running tap
water.
Soak the slide with 95% alcohol and let stand for 30 seconds. Wash it under running tap water.
Soak the slide with safranine and let stand for 30 seconds. Wash it under running tap water.
Dry the slide with tissue paper and add a drop of oil, moreover, examine the bacterium under immersionobjective.
Remember to be accurate with the time.

19

Appendix VI
API test
Dissolve two or three colonies from presumptive isolates in 5 ml API Suspension Medium(free from
additives and chemical compounds e.g Cl2, CO2 etc.).

Distribute the bacterial suspension to all tubes by pipetting. (tilt the strip to avoid bubbles)
For CIT, VP and GEL tests, fill both tube and cupule.
For the other tests, only fill the tube, not the cupule.
For ADH, LDC, ODC, H2S and URE tests, fill cupule with paraffin oil(in order to create anaerobic condition).
Incubate the strip at 37C for 18-24 hours.

TDA test: add 1 drop of TDA reagent and observe the reaction.
IND test: add 1 drop of James reagent and observe the reaction.
VP test: add 1 drop of both VP 1 and VP 2 reagents. Wait for 10 minutes and observe the reaction.
Results were determined by comparing with a positive and negative control strip.

20

Appendix VII
Co-culture of amoeba and bacteria

Streak selected isolates in blood agar medium and incubate at 37C for 16-18 hours.
Transfer two or three colonies to 25 ml LB medium and incubate in 37C for two-three hours.
Perform an absorbance test, if lower than 0.6 keep the sample incubated for a longer time. 0.6
is the required concentration.

Centrifuge 50 ml ATCC medium which contains amoeba at 2000 rpm for 10 minutes.
Count the amoeba under a Brker Chamber (appendix VII) and add 100 l to three separate
wells in a six well plate together with 3 ml ATCC medium.
Add the same amount of bacteria to the three samples containing amoeba.
Do the same procedure with both amoeba in presence and absence of bacteria.

21

Appendix VIII
Counting cells in a Brker Chamber

Mix the sample by pipetting up and down with a 100 l setting.

Soak the slide(Brker chamber) in ethanol(70%) and wipe it with tissue paper. Add the cover
slip.
Transfer 10 l erythrosine B stain to a 1 ml tube and add 10 l from the well(mixed sample),
mix gently by pipetting up and down.
Transfer 10 l to the slide and count the viable (white) cells.

Cells ml-1 was estimated/calculated with the formula:

Where X is the total count of cells and Z is the number of squares counted

22

Egna anteckningar:
ATL Buffer:
AL Buffer:
Proteinase K:
AW1:
AW2:

23