COMPARATIVE ANALYSIS REVEALS A HYPER-VIRULENT ESCHERICHIA COLI O157:H7 STRAIN ISOLATED FROM A SUPER-SHEDDER By LIN TENG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2016
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COMPARATIVE ANALYSIS REVEALS A HYPER-VIRULENT ESCHERICHIA COLI O157:H7 STRAIN ISOLATED FROM A SUPER-SHEDDER
By
LIN TENG
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
Reservoir and Super-Shedder ................................................................................ 18
Role of Super-Shedder ........................................................................................... 19 Factors Contributing to Supper-Shedding ............................................................... 20
Microbial Factors in Super-Shedding ............................................................... 21
Animal Factors Contributing to Super-Shedding .............................................. 25 Environmental Factors Contributing to Super-Shedding .................................. 26
Materials and Methods............................................................................................ 32 Bacterial Strains ............................................................................................... 32 Genomic DNA Extraction .................................................................................. 32 Stx2 Subtyping ................................................................................................. 32
Phage Induction ............................................................................................... 33 Western Blot Analysis of Stx2 Expression ........................................................ 33 Adherence to HEp-2 Cells ................................................................................ 34 Static Biofilm Formation Assay ......................................................................... 35 Genome Sequencing and Assembly ................................................................ 36 Annotation and Comparative Genomics ........................................................... 36
7
In silico Analysis of LSPA-6 Profile and Phylogenetic Clade ............................ 37 In silico Analysis of IS629 Profile ...................................................................... 38
Shiga Toxin Expression of KCJ1266 ................................................................ 39 Adherence to Epithelial Cell Line ...................................................................... 39 Biofilm Formation ............................................................................................. 40
Lineage Determination Based on LSPA-6 ........................................................ 40 Clade of KCJ1266 ............................................................................................ 40 IS629 Profile Distribution .................................................................................. 41
Overview of KCJ1266 Genome ........................................................................ 41
Comparative Genomic Analyses. ..................................................................... 42 Functional Characterization of the Genome ..................................................... 43 Single Nucleotide Polymorphisms .................................................................... 44
Virulence and Colonization Factors .................................................................. 44
Phage Regions in KCJ1266 ............................................................................. 46 Phylogenetic Analysis ...................................................................................... 47
3-3 Genome encoded in the 19 phage regions of KCJ1266 .................................... 54
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LIST OF FIGURES
Figure page 3-1 Shiga-toxin II detection.. ..................................................................................... 56
3-2 Adhesion of E. coli O157:H7 isolates to Hep-2 cells ........................................... 57
3-3 Biofilm assay of KCJ1266. EDL933 was used as a positive control and ΔfimH
mutant of EDL933 was used as a negative control............................................. 58
3-4 IS629 profile of KCJ1266, SS17, EC4115 and EDL933.. ................................... 59
3-5 Genome map of KCJ1266 .................................................................................. 60
3-6 Mauve alignments of KCJ1266 with the three reference genome. ..................... 61
3-7 Comparison of O157 plasmids of KCJ1266 and other 3 reference strains ......... 62
3-8 Subsystem category distribution ......................................................................... 63
3-9 Phage comparison of KCJ1266 with reference strains ....................................... 64
3-10 Virulence related genes in KCJ1266 and reference strains. ............................... 65
3-11 Strains are clustered into three groups which belong to Lineage I (green), Lineage II (red) and Lineage I/II (blue). .............................................................. 66
Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science
COMPARATIVE ANALYSIS REVEALS A HYPER-VIRULENT ESCHERICHIA COLI O157:H7 STRAIN ISOLATED FROM A SUPER-SHEDDER
Figure 3-1. Shiga-toxin II detection. A. Phage induction was measured. MMC was add to cell culture of EDL933,
KCJ1266 and DH5α at the 3rd, 3rd and 4th hour, respectively (Red arrows). B. PCR of stx2a and stx2c C. SDS-PAGE analysis of phage proteins. D. Western blot.
57
Figure 3-2. Adhesion of E. coli O157:H7 isolates to Hep-2 cells, MOI: 100. The Assay was performed in triplicate.
58
Figure 3-3. Biofilm assay of KCJ1266. EDL933 was used as a positive control and ΔfimH mutant of EDL933 was used as
a negative control
59
Figure 3-4. IS629 profile of KCJ1266, SS17, EC4115 and EDL933. A shaded gray box indicates the presence of the in
silico amplicon with correct size and locus, while an unshaded white box indicates a negative in silico result. The three reference strains (EDL933, EC4115 and SS17) are compared.
Str
ain
stx
1
stx
2a
(wrb
A)
stx
2a
(ye
hV
)
stx
2a
(arg
W)
stx
2c
Cla
de
LS
PA
-6
pro
file
Lin
ea
ge
LI-
US
-8
LI-
DS
-17
LI-
US
-9
LI-
DS
-11
LI-
DS
-18
LI-
DS
-1
LI-
DS
-7
LI-
US
-16
LI-
DS
-10
LI-
DS
-19
LI-
DS
-5
LI-
DS
-15
LI-
DS
-6
LI-
US
-13
LI-
DS
-14
LI/
II-D
S-1
5
LI/
II-D
S-4
LI/
II-D
S-7
LI/
II-D
S-1
0
LI/
II-D
S-1
2
LI/
II-D
S-1
3
LI/
II-D
S-1
6
LI/
II-D
S-1
8
LI/
II-D
S-3
LII
-DS
-11
LII
-DS
-3
LII
-DS
-2
LII
-DS
-8
LII
-US
-1
LII
-DS
-10
LII
-DS
-6
LII
-DS
-9
EDL933 3 111111 I
EC4115 8 211111 I/II
SS17 8 211111 I/II
KCJ1266 8 211111 I/II
* * * * * * * * * * * * * * * * * * *
60
Figure 3-5. Genome map of KCJ1266. Marked characteristics are shown from outside to the center. KCJ1266: CDS on
forward strand, tRNA and rRNA in forward strand, tRNA and rRNA in reward strand, CDS on reverse strand, GC content and GC skew; pO157: CDS on forward strand, tRNA and rRNA in forward strand, tRNA and rRNA in reward strand, CDS on reverse strand, GC content and GC skew.
61
Figure 3-6. Mauve alignments of KCJ1266 with the three reference genome. Alignment of KCJ1266 with the three
reference strains (SS17, EC4115 and EDL933) reveal 7 blocks of homology each of varying from ~45.4 kb to ~ 2,276 kb. Each block is a different color with lines connecting corresponding homologous blocks, with the white regions indicating non-homology. (* indicates the block that only exists in SS17 and EDL933)
62
Figure 3-7. Comparison of O157 plasmids of KCJ1266 and other 3 reference strains
63
Figure 3-8. Subsystem category distribution. The subsystem contains 56% of total genes. The other 46% of genes don’t
belong to any of the subsystem.
64
Figure 3-9. Phage comparison of KCJ1266 with reference strains. From inner ring: phage regions of EC4115, phage regions of SS17, phage regions of KCJ1266. The same color represent homologous regions.
65
Figure 3-10. Virulence related genes in KCJ1266 and reference strains. A. Known genes detected in KCJ1266 B. Putative
genes, hypothetical genes, mobile genes and phage related genes detected in KCJ1266
66
Figure 3-11. Strains are clustered into three groups which belong to Lineage I (green), Lineage II (red) and Lineage I/II
(blue). Within Lineage I/II, there are three subgroups which are cluster1, cluster2 and outgroup. KCJ1266, EC4115 and SS17 belong to outgroup of Lineage I/II.
67
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BIOGRAPHICAL SKETCH
Lin Teng was born in Fuzhou, Fujian, China in 1990. He enrolled at the
Huazhong Agricultural University in Wuhan, China, one of the most foremost
Agricultural Universities in China in 2008. His B.S. project focused on constructing
deletion mutant strains of extraintestinal pathogenic Escherichia coli and evaluating the
change of toxicity of mutant strains. Lin earned his Bachelor of Agronomy degree at
Huazhong Agricultural University in 2012. After graduation from the university, Lin
served in Fujian Fengze Group Cooperation, a feed and meat producer, for one year as
a research assistant. In 2014, he was accepted by the Department of Animal Science of
University of Florida as a M.S. student and joined Dr. KwangCheol Casey Jeong’s lab.
During his M.S. program, he got scientific training and participated in some other project,
such as the projects of Chitosan nanoparticles and antimicrobial resistance. His long-
term plan is to work in the industry in China. His immediate plan is to learn more
bioinformatics techniques in the current lab and finish his other ongoing projects.