This slides consists of the result of isolation of bacteriophages against Salmonella Typhi and the evaluation of Salmonella Typhi in bioinformatics method which is online based.

1. Isolation and Evaluation of
bacteriophages against Salmonella Typhi
Prepared by :
Shubashini A/P Balakrishnan (S19080678)
Supervised by :
DR. Lee Su Yin
Bachelor of Science
(HONS) Biotechnology
1

2. Presentation Outline :
1.0
INTRODUCTION
METHODOLOGY
2.0
CONCLUSION
5.0
FUTURE
STUDIES
6.0
RESULT
3.0
DISCUSSION
4.0
2

3. 1.0 INTRODUCTION
3

4. 4
● The term "bacteria eater" is
derived from Greek.
● Bacteriophages are
ubiquitous in the biosphere
● The lysogenic and lytic life
cycle.
● D'Herelle proposed the
term bacteriophage
(Sulakvelidze et al., 2001).
Bacteriophage Salmonella Typhi
● Gram-negative,rod-
shaped and flagellated
bacteria.
● Concerns bacteria for
typhoid fever.
● S.Typhi do not have an
animal or environmental
reservoir.
Antimicrobial resistance
● Can develop in
bacteria, either gram-
positive or gram-
negative
● The three common
mechanisms of
antimicrobial resistance
1.0 INTRODUCTION

5. 1.3 OBJECTIVES
1. To isolate and enrich bacteriophages against Salmonella Typhi from
different environmental samples.
2. To identify the antimicrobial resistance genes (AMR) for Salmonella Typhi.
3. To design specific primers for detection of the AMR genes.
4. To perform in silico evaluation of the specificity of the primers.
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6. 2.0 METHODOLOGY
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7. 2.0 METHODOLOGY
Sample Collection
(15 environment
samples)
Culturing Salmonella
Typhi
01
02
03
Flowchart of an overview of lab-based experiment 7
Isolation of
bacteriophage
(Spot test)

8. 2.0 METHODOLOGY
Identifying
antimicrobial resistance
genes (AMR) for
Salmonella Typhi
Primer designing
Multiple sequence
alignment of strain for
the genes
In silico evaluation of
the primer sequences
01
02 03
04
Flowchart of an overview of online-based
experiment(bioinformatics method)
Blastn the Salmonella
Typhi strain of the
genes
05
8

9. 3.0 RESULT
9

10. 3.2Culturing Salmonella Typhi
Figure 1 : Colonies of S.Typhi on
LB Agar and SS Agar
Figure 2 : Overnight culture
of S.Typhi in LB broth. 10

11. 15 environment
samples were collected
in the sterile plastic
bottles and kept in 4℃
overnight
Filter the water
samples to remove
the solid particles
Then, the filtered
water samples filter
through 0.45μm
followed by a 0.2μm
membrane filter
30ml of filtrate
phage kept inside
the falcon tube
3.2 Isolation of bacteriophages
11

12. Figures 3 : The final result of
Phage enrichment
Figure 4 : The negative spot test on
Salmonella Typhi bacterial lawn
3.3 Isolation of bacteriophages Cont.
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13. 13
Cluny River, Perak
Drain water, SR
Drain Water, Tm
AIMST sewage
Semeling, Tupah, SP
Semeling
Market sample, SR
Merbok river, SP
Market sample, SP
Sg.Bil river, Sr
Sewage sample, SR
Sewage sample, TM

14. 3.3 AMR genes for Salmonella Typhi
Figure 5: The result of AMR genes of Salmonella Typhi
Which retrieved from :MicroBIGG-E,NDARO. There are 19875 AMR genes. 14

15. 3.3 AMR genes for Salmonella Typhi
Figure 6 : The three AMR genes of Salmonella Typhi
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16. Figure 10 :The multiple sequence results of ten
different sequences of Salmonella Typhi of gyrA
gene.
Figure 11 :The multiple sequence alignment
results of ten different strains of Salmonella Typhi
of Sul1 gene.
3.4 Multiple sequence alignment (MSA) using Clustal Omega
16

17. Figure 12 : The multiple sequence alignment results of
ten different strains of Salmonella Typhi of Sul2 gene.
3.4 Multiple sequence alignment (MSA) using Clustal Omega
17

18. Figure 13 :Forward and reverse primers of gyrA gene
Figure 15 : Forward and reverse primers of Sul2 gene
Figure 14 : Forward and reverse primers of Sul1 gene
3.5 Primer designing for detection of the AMR genes.
18

19. Figure 29 : The blast result of the forward
and reverse primers (Sul1)
Figure 28 : The blast result of the
forward and reverse primers (gyrA)
3.5 Primer designing for detection of the AMR genes
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20. Figure 30 : The blast result of the forward and reverse primers (Sul2)
3.5 Primer designing for detection of the AMR genes
20

21. The specificity of 10
positive and negative sense
sequences are 60% and 40%
to each other forward and
reverse primers, respectively.
Figure 31 : The stratify result of forward primer (gyrA)
Figure 32 : The stratify result of reverse primer (gyrA)
3.6 in silico evaluation of the primers
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22. The specificity of 15 positive
sense sequences are 100% to each
other forward primers and also
100% specificity for reverse
primers.
Figure 34 :The stratify result of forward primer (Sul1)
Figure 35 : The stratify result of reverse primer (Sul1)
3.6 in silico evaluation of the primers
22

23. The specificity of 10 positive
and negative sense sequences
are 66.67% and 33.33% to
each other forward and
reverse primers, respectively.
Figure 37: The stratify result of forward primer (Sul2)
Figure 38: The stratify result of reverse primer (Sul2)
3.6 in silico evaluation of the primers
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24. 4.0 DISCUSSION
24

25. ● Initially, spot testing was conducted.
● Spot test result: Negative
● This method need more phage concentration because many samples
do not contain sufficient quantities of phages.
.
● Isolate the phages by doing the enrichment cultures (Culture lysis)
● Clear zone : None
● This result is due to the factor affect the survival and number of
phages in the water sources include the temperature, radiation, pH
value and presence of chemicals.
3.3 Isolation of bacteriophages Cont.
25

26. Factors affect the presence of
phage
Explanation
pH value ● Phages survive well within the pH limits.
● Inactive in water sources with pH values between 5 to 9.
Radiation ● Direct sun radiation is also deleterious to phage survival.
● Thermal inactivation of phages is generally considered
exponential.
Presence of chemicals ● Urea and urethane could inactivate phages
● Besides, many detergents can inactivate phages even though
the effect differs from phage to phage
4.1 Isolation of bacteriophage
26

27. QUERY COVER
(%)
● A number that describes how much of the query
sequence is covered by the target sequence
● ↑ Q.cover, indicates that sequence overlapped
matched very well.
● ↓ Q.cover indicates that the overlap with the reference
sequence is very low
PERCENTAGE
IDENTITY (%)
● % identity is the percentage of residues that
match up in the alignment.
● ↑ percent identity is, the more significant the
match.
4.2 AMR genes similarity between other Salmonella Typhi strains Using BLAST
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28. 4.2 Multiple Sequence Alignment using Clustal Omega
1. The percent identity matrix for 3 genes are 100%
2. High homology sequences resulting from CLUSTAL Omega's pruning of
previously matched sequences.
3. High homology means there are no gaps.
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29. 4.4 Primer designing for detection of the AMR genes.
1. All the primers were designed to fit the factors of a primer.
2. The forward and reverse primer oligonucleotides were between 18 to 24 base pairs
or nucleotides.
3. The GC content of all the forward and reverse primers is between 40% to 60%.
4. In addition, the delta G should be near zero value
5. The annealing temperature must be lower than the Tm of the primers
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30. 3.5 in silico evaluation of the primers
1. To examine the ability of primers to detect a broad taxonomic range of arthropods,
regardless of sample quality and laboratory techniques (extraction and PCR ).
2. In addition, the primer pair of gyrA and Sul1 demonstrated a high level of PCR
efficiency.
3. Consequently, the low effectiveness of the inverted primer Sul2 may be connected
with high degeneracy at particular locations of the oligonucleotide.
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31. 5.0 CONCLUSION
31

32. Conclusion
● This study is about to isolate and enrich the bacteriophage against Salmonella Typhi
● Unfortunately, the phage against Salmonella Typhi could not be successfully
obtained whereas successfully identified the AMR genes of Salmonella Typhi and
evaluate all the three genes.
● With these results, these studies achieved all the objectives and successfully
proposed all the primers for further studies.
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33. 6.0 FUTURE
STUDIES
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34. 6.0 FUTURE STUDIES
● Clinical samples, poultry samples and hospital
sewage
● The reason of suggesting these samples :
Stool
samples
Densely populated with microbes
including enormous amounts of phages
Poultry
samples
Hospital sewages only receive human
waste from hospital
Hospital
sewage
The intended host, Salmonella Typhi,
can be present in all of these sources
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35. REFERENCES
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37. THANK YOU !
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