Nykpingsgymnasium 17/4-13 Natural sciences programme

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

Acanthamoeba castellanii

show poor symbiosis with

bacterial Spp from estaurine

environments in Nykping

2

Abstract

Acanthamoebae castellanii serves as an environmental host for bacterial spp during inconvenienc-

es. Amoebae enhance the surviving skills of bacterial spp. Furthermore, the impact of global warm-

ing on water temperature leads to bacterial rates and increased infections caused by diverse bacte-

ria. 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 identifica-

tion 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 Nyk-

ping and investigate if they lived symbiotically with bacterial spp.

3

Table of content

1. Introduction 4

1.1. Background 4

1.2. Purpose and aim 4

1.3. Question formulation 4

1.4. Limitations 4

1.5. Factual background 5

2. Material and Methods 6

2.1. Reading materials 6

2.1.1. Sample collection 6

2.1.2. DNA extraction and PCR 6

2.1.3. Gel analysis of PCR product 6

2.2. Isolation and identification of microbals 6

2.2.1. Gram staining 6

2.2.2. Biochemical identification of selected isolates 6

2.2.3. Catalase, DNase and oxidase 6

2.2.4. Amoeba culture 7

2.3. Bacterial strains 7

2.3.1. Culture media, growth conditions and analysis 7

2.3.2. Microscopy analysis 7

2.4. Limitations 7

3. Results 7

3.1. Gel electrophoresis 7

3.2. API test 7

3.3. Amoebaculture 8

3.4. Catalase, DNase and oxidase 8

3.5. Growth of A.castellani 8

4. Discussion 9-12

5. Acknowledgements 12

6. References 13-14

7. Appendix 15

7.1. Appendix I 15

7.2. Appendix II 16

7.3. Appendix III 17

7.4. Appendix IV 18

7.5. Appendix V 19

7.6. Appendix VI 20

7.7. Appendix VII 21

7.8. Appendix VIII 22

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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 laborato-

ry 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 Mem-

brane 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 Nyk-

ping (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 signifi-

cantly. That means that bacterial growth in aquatic environments is benefitted both from global warm-

ing 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 en-

hance 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 tem-

perature, 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.

5

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 spe-

cies are capable of surviving and multiplying inside amoebae(7,10-12). Furthermore, amoebae con-

taining bacteria can cause infections and these infections occur worldwide (7-12). Acanthamoeba cas-

tellanii 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, mo-

tile 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 condi-

menti(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 37-

43C.(2-4). Numerous studies disclose the OmpA as a contributory virulence factor. OmpA in Crono-

bacter species aids the infection by facilitating the penetration of the blood barrier, furthermore, induc-

ing 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 uti-

lizes biofilm formation which facilitates its survival and it produces an enterotoxin, but, the im-

portance 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 molecu-

lar 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 in-

6

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 spe-

cies 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 mi-

crobiology books.

2.1.2 Sample collection Four water samples of 1.2 L each was collected from the river of Nykping (Nykpingsn). The col-

lection 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 dis-

carded. The same procedure was performed with all water samples and the pellet from the same col-

lection 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 meth-

od, 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.

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.

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 iso-

late(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 micro-

scope.

2.3 Bacterial strains Bacterial strains used in the interaction study are Enterobacter sakazakii(Cronobacter Spp) and Bacil-

lus 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 10

6 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 Ap-

pendix 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 lac-

tose fermentation.

3. Results

3.1 Gel electrophoresis The result showed that all samples contained Acan-

thamoeba 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 iso-

lates as Enterobacter sakazakii (Cronobacter

cies) with 98.4% coincidence, and as Vibrio

8

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).

lyticus with 72% accura-

cy. Remaining isolates

could not be determined

by the API 20E test.

3.3 Amoeba culture 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.

*Estimated species, according to type schedule for aerobic and facultative anaerobic bacteria.

Source/isolate Colonial morphology Gram

Stain

Morphology Species* Additional test

Piren 1 Small white G- Rod+long chain

Piren 2 Large white G- Rod Enterobacter sakazakii(98.4%) API

Piren 3 Large yellow G+ Rod+diplococcus Micrococcus Spp

ngstugan 1 Small white G- Rod Negative test API

ngstugan 2 Large pale(hemo) G+ Rod Bacillus subtilis

ngstugan 3 Large yellow G+ Diplococcus Micrococcus Spp

ngstugan 4 Large white G- Rod(small) Pseudomonadaceae Oxidase

ngstugan 5 Large pale G+ Diplococcus Micrococcus Spp

Tjuvholmen 1 Small white G- Rod

Tjuvholmen 2 Medium pale(hemo) G- Rod+long chain Vibrio alginolyticus (72.4%) API

Tjuvholmen 3 Large white G+ Rod Bacillus subtilis

Tjuvholmen 4 Large yellow G+ Diplococcus Micrococcus Spp

Tjuvholmen 5 Medium white G+ Coccus Staphylococcus epidermidis Catalase, DNase

Hllet 1 Small white G- Rod+long chain

Hllet 2 Medium white G- Rod Negative test API

Hllet 3 Large yellow G+ Rod Bacillus subtilis

9

Discussion

The information which was used in this study is acquired from scientific articles, mainly from Pub-

Med. PubMed is a database with both recent and old articles on research in medical and biological sci-

ence. The information acquired is verified by reading different articles supporting the same infor-

mation.

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 per-

formed. The gel electrophoresis is a good test since it is not very likely that it may become contami-

nated, and, if properly performed one obtains good results.

The typing schedule which was used for the bacterial identification was acquired from Mamun Ur Ra-

shid. 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 com-

plement 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 Pseudomona-

daceae not Pseudomonas aeruginosa.

All samples were vortexed and grown on both blood and CLED agar plates. Blood agar is a rich medi-

um 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 per-

formed 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 ac-

cording 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 in-

teraction 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.

10

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. algino-

lyticus, 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 de-

termined 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 ex-

amination is a type of mass spectrometry, where the bacterium is determined by ionizing of a particu-

lar compound. The bacterial identification may also be performed as I have done it, according to a typ-

ing 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 au-

reus 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 look-

ing 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 amoe-

bae or not. This assay includes isolation of amoeba, gentamycin(antibiotic) treatment, growth of extra-

cellular and intracellular bacteria and count of observed colonies.

The extensive methodology of this project should be considered while performing it. The time plan-

ning 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 con-

tinuous way of working. The budget should neither be restricted as in my case in terms of the addi-

tional planned assays.

11

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 pos-

sible 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. Howev-

er, the isolates from Piren 2 and Tjuvholmen 2 showed positive results and were identified as Entero-

bacter 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 ex-

plained.

In addition, the high presence of V.alginolyticus is interesting. Two of the articles that I have read sug-

gests an emerging increase in Vibrios, leading to an increase in infections caused by Vibrios. The pres-

ence 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 ver-

ify 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 amoe-

bae 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 symbi-

otic effect. A prediction can be that the amoebae may not be strong or satisfied with the nutrient, fur-

ther 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 typ-

ing 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 in-

hibit the growth of A.castellanii with exoenzymes(13). This means that the extensive production of

12

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 rea-

son. 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 es-

timated by Mamun Ur Rashid who is a bacteriologist. The estimated species need to be further con-

firmed 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 per-

form 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 coun-

ty for letting me analyze the water and providing me with travelling costs.

13

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15

Appendix I Map of Nykping

Map over the sample collection sites. 1.2 L of samples was obtained from each Hllet, Piren, ng-

stugan and Tjuvholmen. Sample location sites are displayed with a star.

16

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 col-umn 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

Primer 2

1.5l

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 tem-

plates.

Place the PCR tubes in the thermal cycler and start the cycling programe.

Cycling conditions

Step Time Temperature

Initial heat activation 15 min 95C

Denaturation 1 min 94C

Annealing 1 min 50C

Extension 1 min 72C

Number of cycles(20) 30

Final extension 10 min 72C

17

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.

18

19

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 immer-

sionobjective.

Remember to be accurate with the time.

20

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 an-aerobic 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.

21

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.

22

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

23

Egna anteckningar:

ATL Buffer:

AL Buffer:

Proteinase K:

AW1:

AW2: