# Genotype Cluster Analysis in PathogenicEscherichia coli Isolates Producing Different CDT Types

Authors: Maryam Javadi; Mana Oloomi; Saeid Bouzari
Journal: Journal of Pathogens
 (2016)

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## Abstract

Diarrheagenic and uropathogenicE. coli types are mainly characterized by the expression of distinctive bacterial virulent factors.stx1,stx2 (Shiga toxins), andcdt (cytolethal distending toxin) genes have been acquired by horizontal gene transfer. Some virulent genes such asespP (serine protease),etpD (part of secretion pathway), andkatP (catalase-peroxidase), orsfpA gene (Sfp fimbriae), are on plasmids and the others likefliC(flagellin) and thefimH gene (fimbriae type-I) are located on chromosome. Genomic pathogenicity islands (PAIs) carry some virulent genes such ashly gene. To determine the existence of virulence genes incdt clinical isolates, genes includingstx1,stx2,cdt,hly,espP,katP,sfpA,etpD,fliC, andfimH were assessed by Polymerase Chain Reaction (PCR). The most prevalent isolates foretpD andkatP genes were 85.7% incdtII.katP gene was also observed 83.3% incdtI. However, in 42.85% ofcdtIII isolates,espP gene was the most detected. Moreover,hly gene was also the most prominent gene incdtIII (71.42%).sfpA gene was observed in 66.6% ofcdtV.stx1 gene was detected in 100% ofcdtII,cdtIV, andcdtV types. Presence and pattern of virulence genes were considered amongcdt positive isotypes and used for their clustering and profiling.


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## Body

## 1. Introduction

Escherichia coli  (E. coli) are known as a commonly colonizing bacteria of the human intestinal tract. Foreign DNA including plasmids, pathogenicity islands (PAIs), transposons, and phages are acquired through the horizontal gene transfer by an ancient nonpathogenicE. coli strain and led to development of specific pathotypes. However, the genomic background has a pivotal role in evolutionary pathways. PathogenicE. coli strains are classified based on repertoires of virulence factors and the most common diseases associated with them.Shiga toxins are a family of related toxins with two major groups, Stx1 and Stx2, expressed by genes considered to be horizontally acquired by bacteriophages. Shiga toxin encoded bystx1 andstx2 genes is an A-B type toxin that inhibits protein synthesis and causes hemorrhagic colitis and hemolytic-uremic syndrome. Thestx genes are located in the genome of heterogeneous lytic (stx2) or cryptic (stx1) lambdoid phages [1–4]. Cytolethal distending toxins (CDTs) were the firstly recognized bacterial toxins that block the eukaryotic cell cycle, suppress cell proliferation, and eventually lead to cell death. A CDT is a tripartite holotoxin in whichcdtB is the active subunit and has DNAase-I-like activity [5]. Five different CDTs (I–V) have been reported forE. coli so far, and they were designated in order of publications. CDT production has been associated with pathogenicE. coli. The presence ofcdt genes in different bacterial species and the analysis of the DNA in the vicinity of thecdt genes suggest that the toxin has been acquired from heterogenic species by horizontal gene transfer. However, the probable phylogenetic origin (or ancestor) has still remained elusive. Interestingly, the phage and the corresponding insertion sequence remnants were found nearby theE. coli cdt genes. These data suggest thatcdt genes were acquired by horizontal transfer events at some point and evolved separately since then [5, 6].As withstx gene, some types ofcdt genes are horizontally acquired by phages inE. coli [6, 7].It is now evident that some virulent genes are located on a large virulent plasmid (pO157) in pathogenicE. coli, among which are the extracellular serine protease gene (espP), catalase-peroxidase gene (katP), and type II secretion pathway protein D (etpD). Besides different sizes of this plasmid were reported and may not contain all these 3 genes which indicate independency of genetic exchanges through the horizontal gene transfer process. EspP can be grouped into the autotransporter proteins family and characterized by catalytically active serine residue in the active center. EspP cleaves pepsin and human coagulation factor V [8–11]. Catalase-peroxidase gene (katP) encodes a protein which shows bifunctional catalase and peroxidase activity [12]. This enzyme is expressed by pathogenic strains and has been thought to protect these pathogens from oxidative damage caused by reactive oxygen molecules produced by phagocytes or other host cells during the infection process [13]. Type II secretion pathway protein D encoded byetpD gene is another pathogenic factor encoded on the aforementioned plasmid [14].A cluster of six genes, termedsfp includingsfpA gene, are located on another plasmid, pSFO157, in some pathogenicE. coli strains. This genomic cluster mediates mannose-resistant hemagglutination and expression of a novel type of fimbriae, Sfp fimbriae, which is 3–5 nm in diameter, the major subunit of which is SfpA [15, 16].Type 1 fimbriae, encoded by a chromosomally locatedfim gene cluster, are the most common adhesive organelles ofEscherichia coli. Fimbriae-mediated adherence, which facilitates colonization and survival in host cells, plays a significant role in pathogenesis. Type 1 fimbriae consist of a major structural subunit (FimA) and several minor components, including adhesin (FimH) [17, 18].fliC gene encoding the flagellin subunit is located on chromosome and could be considered as genomic background. Flagella facilitate bacterial movement and have a vital role in bacterial distribution in intestine and host tissues [19].Pathogenicity islands (PAIs) are a subgroup of genomic islands that carry one or more virulent genes and are present in the genome of a pathogenic bacterium but absent from the genomes of nonpathogenic species. PAIs occupy relatively large genomic regions. The regions carrying hemolysin gene (hly) is located in PAI ofE. coli chromosome [20–22].In this study, the occurrence of virulent plasmid-borne genes, pathogenicity island, and chromosomally encoded genes in CDT-producingE. coli strains was investigated.


## 2. Materials and Methods

### 2.1. Bacterial Strains

In this study, 30 CDT-producing strains were investigated. The strains were isolated from clinical diarrheal patients [25, 26] and were cultured overnight at 37°C in Luria Bertani (LB) medium.


### 2.2. DNA Extraction

Template DNA extraction was performed by Phenol-Chloroform assay (1 mL from cultured LB centrifuged at 14000 rpm for 3 minute), and then the pellet was dissolved in 1 mL of 5 mM Tris-HCl pH = 6.8, centrifuged at 10000 rpm for 2 minutes. The pellet was then resuspended in 350μL of 5 mM Tris-HCl pH = 6.8 with 25 μL lysozyme enzyme (5 mg/mL) and 25 μL of 1 M Tris-HCl, pH: 8.8, and incubated for 15 minutes in room temperature. Next, 15 μL 0.5 M EDTA was added with 40 μL SDS10% and 10 μL RNase enzyme (20 mg/mL), mixed gently, and treated at 37°C for 2-3 hours. After adding 5 μL proteinase K (20 mg/mL), the samples were incubated overnight at 37°C and, afterwards, heated for 5 minutes at 55°C. Then, 400 μL phenol was added and mixed gently and 400 μL chloroform was added, mixed vigorously or by vortex, and centrifuged for 20 minutes (14000 rpm). The upper phase was collected, and 800 μL cold ethanol 96% was added and left at room temperature for 15–30 minutes and then was centrifuged at 14000 rpm for 15 minutes. The upper phase was discarded and the pellet was washed 1-2 times, dried, and dissolved in 50 μL TE (1x) and then heated at 75°C for 15 minutes.In addition, elution was performed using TE (1x) buffer. Totally, the concentration of templates was adjusted to 100 ng/μL.


### 2.3. Amplification of Target Genes and PCR Conditions

Virulence-associated genes, includingfliC,fimH,stx1,stx2,etpD,espP,sfpA,katP, andhly genes, were assessed by Polymerase Chain Reaction (PCR).cdt typing based on specific primers was performed. In Table 1 the nucleotide sequence of primers used for amplification of target genes is depicted.Table 1
Nucleotide sequence of primers for amplification of target genes.
Target gene
Nucleotide sequence (forward/reverse)
Size (bp)
Reference
sfpA
5′-AGC CAA GGC CAA GGG ATT ATT A-3′ 5′-TTA GCA ACA GCA GTG AAG TCT C-3′
440-bp
[23]
espP
5′-AAA CAG CAG GCA CTT GAA CG-3′ 5′-GGA GTC GTC AGT CAG TAG AT-3′
1830-bp
[24]
katP
5′-CTT CCT GTT CTG CTG ATT CTT CTG G-3′ 5′-AAC TTA TTT CTC GCA TCA TCC-3′
2125-bp
[24]
etpD
5′-CGT CAG GAG GAT GTT CAG-3′ 5′-CGA CTG CAC CTG TTC CTG ATT A-3′
1062-bp
[24]
fliC
5′-CCA GTC ATT AAT ACA AAC AGC C-3′ 5′-GAC ATA TTA GAG ACT TCG GT-3′
1600-bp
[23]
fimH
5′-CTT ATG GCG GCG TGT TAT CTA-3′ 5′-GCC AGT AGG CAC TAC CAC ATC-3′
289-bp
[1]
hly
5′-AGG TTC TTG GGC ATG TAT CCT-3′ 5′-TTG CTT TGC AGA CTG CAG TGT-3′
555-bp
[25]
stx1
5′-CGA TGT TAC GGT TTG TTA CTG TGA CAG C-3′ 5′-AAT GCC ACG CTT CCC AGA ATT G-3′
244-bp
[25]
stx2
5′-GTT TTG ACC ATC TTC GTC TGA TTA TTG AG-3′ 5′-AGC GTA AGG CTT CTG CTG TGA C-3′
324-bp
[25]
cdt-I
5′-CAA TAG TCG CCC ACA GGA-3′ 5′-ATA ATC AAG AAC ACC ACC AC-3′
411-bp
[26]
cdt-II
5′-GAA AGT AAA TGG AAT ATA AAT GTC CG-3′ 5′-TTT GTG TTG CCG CCG CTG GTG AAA-3′
556-bp
[26]
cdt-III
5′-GAA AGT AAA TGG AAT ATA AAT GTC CG-3′ 5′-TTT GTG TCG GTG CAG CAG GGA AAA-3′
555-bp
[26]
cdt-IV
5′-CCT GAT GGT TCA GGA GGC TGG TTC-3′ 5′-TTG CTC CAG AAT CTA TAC CT-3′
350-bp
[26]
cdt-V
5′-AGC ACC CGC AGT ATC TTT GA-3′ 5′-AGC CTC TTT TAT CGT CTG GA-3′
1363-bp
[26]PCR samples were prepared in a total volume of 25μL (PCR buffer: (10x) buffer/CinnaGen Co, dNTP: 25 mM/Fermentas, Taq DNA polymerase: 5 units per μL/CinnaGen Co, MgCl2: 50 mM/CinnaGen Co., D.D.W., Template: extracted genomic DNA (100 ng/μL), Thermocycler: SENSOQUEST Labcycler).


### 2.4. Cloning and Sequencing

In this study, we cloned some positive samples and sequenced the PCR products. After PCR optimization and gene amplification, gene extraction from gel agarose was done with “Core one™ Gel extraction kit GE-100, CoreBioSystem Co. Ltd.” We also utilized pTZ57R/T vector for ligation process which was done with InsTA clone™ PCR Cloning Kit (Fermentas). Recombinant cells were cultured on plates containing Ampicillin (500 mg/mL).


### 2.5. Hierarchical Clustering

Virulence-associated genes of strains were compared based on dendogram illustration. A hierarchical clustering analysis was performed and dendrogram was constructed by IBM SPSS Statistics software (Version 20). All virulent genes regarded as variables and statistics setting was set on Agglomeration schedule. Cluster method was between-groups linkage by measuring squared Euclidean distance.


## 2.1. Bacterial Strains

In this study, 30 CDT-producing strains were investigated. The strains were isolated from clinical diarrheal patients [25, 26] and were cultured overnight at 37°C in Luria Bertani (LB) medium.


## 2.2. DNA Extraction

Template DNA extraction was performed by Phenol-Chloroform assay (1 mL from cultured LB centrifuged at 14000 rpm for 3 minute), and then the pellet was dissolved in 1 mL of 5 mM Tris-HCl pH = 6.8, centrifuged at 10000 rpm for 2 minutes. The pellet was then resuspended in 350μL of 5 mM Tris-HCl pH = 6.8 with 25 μL lysozyme enzyme (5 mg/mL) and 25 μL of 1 M Tris-HCl, pH: 8.8, and incubated for 15 minutes in room temperature. Next, 15 μL 0.5 M EDTA was added with 40 μL SDS10% and 10 μL RNase enzyme (20 mg/mL), mixed gently, and treated at 37°C for 2-3 hours. After adding 5 μL proteinase K (20 mg/mL), the samples were incubated overnight at 37°C and, afterwards, heated for 5 minutes at 55°C. Then, 400 μL phenol was added and mixed gently and 400 μL chloroform was added, mixed vigorously or by vortex, and centrifuged for 20 minutes (14000 rpm). The upper phase was collected, and 800 μL cold ethanol 96% was added and left at room temperature for 15–30 minutes and then was centrifuged at 14000 rpm for 15 minutes. The upper phase was discarded and the pellet was washed 1-2 times, dried, and dissolved in 50 μL TE (1x) and then heated at 75°C for 15 minutes.In addition, elution was performed using TE (1x) buffer. Totally, the concentration of templates was adjusted to 100 ng/μL.


## 2.3. Amplification of Target Genes and PCR Conditions

Virulence-associated genes, includingfliC,fimH,stx1,stx2,etpD,espP,sfpA,katP, andhly genes, were assessed by Polymerase Chain Reaction (PCR).cdt typing based on specific primers was performed. In Table 1 the nucleotide sequence of primers used for amplification of target genes is depicted.Table 1
Nucleotide sequence of primers for amplification of target genes.
Target gene
Nucleotide sequence (forward/reverse)
Size (bp)
Reference
sfpA
5′-AGC CAA GGC CAA GGG ATT ATT A-3′ 5′-TTA GCA ACA GCA GTG AAG TCT C-3′
440-bp
[23]
espP
5′-AAA CAG CAG GCA CTT GAA CG-3′ 5′-GGA GTC GTC AGT CAG TAG AT-3′
1830-bp
[24]
katP
5′-CTT CCT GTT CTG CTG ATT CTT CTG G-3′ 5′-AAC TTA TTT CTC GCA TCA TCC-3′
2125-bp
[24]
etpD
5′-CGT CAG GAG GAT GTT CAG-3′ 5′-CGA CTG CAC CTG TTC CTG ATT A-3′
1062-bp
[24]
fliC
5′-CCA GTC ATT AAT ACA AAC AGC C-3′ 5′-GAC ATA TTA GAG ACT TCG GT-3′
1600-bp
[23]
fimH
5′-CTT ATG GCG GCG TGT TAT CTA-3′ 5′-GCC AGT AGG CAC TAC CAC ATC-3′
289-bp
[1]
hly
5′-AGG TTC TTG GGC ATG TAT CCT-3′ 5′-TTG CTT TGC AGA CTG CAG TGT-3′
555-bp
[25]
stx1
5′-CGA TGT TAC GGT TTG TTA CTG TGA CAG C-3′ 5′-AAT GCC ACG CTT CCC AGA ATT G-3′
244-bp
[25]
stx2
5′-GTT TTG ACC ATC TTC GTC TGA TTA TTG AG-3′ 5′-AGC GTA AGG CTT CTG CTG TGA C-3′
324-bp
[25]
cdt-I
5′-CAA TAG TCG CCC ACA GGA-3′ 5′-ATA ATC AAG AAC ACC ACC AC-3′
411-bp
[26]
cdt-II
5′-GAA AGT AAA TGG AAT ATA AAT GTC CG-3′ 5′-TTT GTG TTG CCG CCG CTG GTG AAA-3′
556-bp
[26]
cdt-III
5′-GAA AGT AAA TGG AAT ATA AAT GTC CG-3′ 5′-TTT GTG TCG GTG CAG CAG GGA AAA-3′
555-bp
[26]
cdt-IV
5′-CCT GAT GGT TCA GGA GGC TGG TTC-3′ 5′-TTG CTC CAG AAT CTA TAC CT-3′
350-bp
[26]
cdt-V
5′-AGC ACC CGC AGT ATC TTT GA-3′ 5′-AGC CTC TTT TAT CGT CTG GA-3′
1363-bp
[26]PCR samples were prepared in a total volume of 25μL (PCR buffer: (10x) buffer/CinnaGen Co, dNTP: 25 mM/Fermentas, Taq DNA polymerase: 5 units per μL/CinnaGen Co, MgCl2: 50 mM/CinnaGen Co., D.D.W., Template: extracted genomic DNA (100 ng/μL), Thermocycler: SENSOQUEST Labcycler).


## 2.4. Cloning and Sequencing

In this study, we cloned some positive samples and sequenced the PCR products. After PCR optimization and gene amplification, gene extraction from gel agarose was done with “Core one™ Gel extraction kit GE-100, CoreBioSystem Co. Ltd.” We also utilized pTZ57R/T vector for ligation process which was done with InsTA clone™ PCR Cloning Kit (Fermentas). Recombinant cells were cultured on plates containing Ampicillin (500 mg/mL).


## 2.5. Hierarchical Clustering

Virulence-associated genes of strains were compared based on dendogram illustration. A hierarchical clustering analysis was performed and dendrogram was constructed by IBM SPSS Statistics software (Version 20). All virulent genes regarded as variables and statistics setting was set on Agglomeration schedule. Cluster method was between-groups linkage by measuring squared Euclidean distance.


## 3. Results

### 3.1. Virulent Genes Detection

Existence of virulence genes, includingespP,etpD,katP,sfpA,hly,fliC,fimH,stx1,stx2, andcdt genes, was assessed by PCR (Table 2).Table 2
The occurrence of virulence-associated genes including plasmid, Pathogenicity Island, chromosomal and phage genes. (+) indicates positive PCR product and the existence of mentioned gene while (−) indicates negative PCR product.
Strain
espP
etpD
katP
sfpA
Hly
fliC
fimH
cdt-I
cdt-II
cdt-III
cdt-IV
cdt-V
stx1
stx2
1
−
+
+
+
−
+
+
+
−
−
−
−
−
−
2
−
−
−
−
−
−
+
+
−
−
−
−
+
−
3
−
−
+
−
−
+
+
+
−
−
−
−
+
−
4
+
−
+
−
−
+
+
+
−
−
−
−
+
−
5
−
−
+
−
−
+
+
+
−
−
−
−
+
+
6
−
+
+
−
−
+
+
+
−
−
−
−
+
+
7
−
+
+
+
−
+
+
−
+
−
−
−
+
−
8
−
+
+
−
−
+
+
−
+
−
−
−
+
−
9
−
−
+
−
+
+
+
−
+
−
−
−
+
−
10
−
+
+
−
−
+
+
−
+
−
−
−
+
−
11
−
+
+
−
−
+
+
−
+
−
−
−
+
−
12
−
+
+
−
−
+
+
−
+
−
−
−
+
−
13
−
+
−
−
−
+
+
−
+
−
−
−
+
−
14
+
−
+
−
−
+
+
−
−
+
−
−
−
−
15
+
−
+
−
+
+
+
−
−
+
−
−
−
−
16
−
+
+
−
+
+
+
−
−
+
−
−
−
−
17
+
−
−
+
−
+
+
−
−
+
−
−
+
−
18
−
−
+
−
+
+
+
−
−
+
−
−
+
−
19
−
−
−
−
+
+
+
−
−
+
−
−
+
+
20
−
+
−
−
+
+
+
−
−
+
−
−
+
−
21
+
+
+
+
−
+
+
−
−
−
+
−
+
−
22
−
−
+
+
+
+
+
−
−
−
+
−
+
−
23
−
+
−
−
−
+
+
−
−
−
+
−
+
−
24
+
+
+
−
+
+
+
−
−
−
+
−
+
+
25
−
+
−
+
+
+
+
−
−
−
+
−
+
−
26
−
−
−
−
−
+
+
−
−
−
+
−
+
−
27
−
−
+
−
+
+
+
−
−
−
+
−
+
−
28
+
−
−
−
−
+
+
−
−
−
−
+
+
−
29
−
+
−
+
+
+
+
−
−
−
−
+
+
−
30
−
+
−
+
+
+
+
−
−
−
−
+
+
−All strains werecdt positive and regardingcdt types 6 strains werecdt-I (20%), 7 strains (23.3%) werecdt-II,cdt-III, andcdt-IV, and 3 strains were recognized ascdt-V (10%).In the present study,stx1 gene was present in 86.7% of clinical isolates. However, thestx2 gene was detected in 13.3% of strains.stx1 gene was also detected in 83.3% ofcdt-type-I producing strains, while it was detected in all thecdt-typeII,IV,V strains and in 57.1% ofcdt-III isolates (Table 3).stx2 gene was present at 33.3% ofcdt-type I strains, 14.3% incdt-typeIII,IV strains, while in CDT-II and CDT-V producing isolates it was not observed.Table 3
The frequency of target genes in differentcdt-type isolates.
cdt type
Target gene
katP
etpD
espP
sfpA
stx1
stx2
hly
cdt-type I
83.33%
33.33%
16.6%
16.66%
83.33%
33.33%
0%
cdt-type II
85.7%
85.7%
0%
14.28%
100%
0%
14.28%
cdt-type III
57.14%
28.57%
42.85%
14.28%
57.14%
14.28%
71.42%
cdt-type IV
57.14%
57.4%
28.57%
42.85%
100%
14.28%
57.14%
cdt-type V
0%
66.66%
33.33%
66.66%
100%
0%
66.66%hly gene was not detected in any of CDT-I-producing strains. However, it was detected in 14.3% ofcdt-II strains, 71.4% ofcdt-III strains, 57.1% ofcdt-IV strains, and 66.7% of CDT-V-producing isolates (Table 3). In this clinical isolates, 53.3%etpD, 63.3%katP,and 23.3%espPgenes were detected. In the present study,etpD gene was observed in 33.3% ofcdt-type I, 85.7% ofcdt-type II, 28.6% ofcdt-type III, 57.1% ofcdt-type IV, and 66.7% ofcdt-type V strains.espP gene was present in 16.7% ofcdt-type I, 42.9% of CDT-III-producing, 28.6% ofcdt-type IV, 33.3% ofcdt-type V strains, while, in CDT-II-producing isolates, the gene was not detected. In addition,katP gene was detected in 83.3% ofcdt-type I, in 85.7% ofcdt-type II, in 57.1% of CDT-III- and CDT-IV-producing strains, while, this gene was not observed in CDT-V-producing strains (Table 3).Occurrence of virulent genes,espP,etpD,katP, andsfpA, was classified as an A–M pattern which was based on the existence of plasmid encoding genes (Table 4).Table 4
Differentcdt-type strains and related genotype based on other virulent genes and profile designation based on cooccurrence of plasmid-borne genes. espP: profile A, etpD: profile B, katP: profile C, espP/katP: profileD, espP/sfpA: profile E, etpD/katP: profile F, etpD/sfpA: profile G, katP/sfpA: profile H, etpD/katP/sfpA: profile I, espP/etpD/katP: profile K, espP/etpD/katP/sfpA: profile L, and  profile M: Indicates the absence of plasmid genes and presence of chromosomal genes (fimH, fliC, hly, stx1, or stx2).
Strain
cdt-type
Genotype based on virulence-related genes
Profile designation based on plasmid-borne genes
1
cdt-I
fimH,fliC, etpD,katP,sfpA
I
2
cdt-I
fimH,stx1
M
3
cdt-I
fimH,fliC,stx1, katP
C
4
cdt-I
fimH,fliC,stx1, espP,katP
D
5
cdt-I
fimH,fliC,stx1,stx2, katP
C
6
cdt-I
fimH,fliC,stx1,stx2, etpD,katP
F
7
cdt-II
fimH,fliC,stx1,etpD,katP,sfpA
I
8
cdt-II
fimH,fliC,stx1,etpD,katP
F
9
cdt-II
fimH,fliC,hly,stx1,katP
C
10
cdt-II
fimH,fliC,stx1,etpD,katP
F
11
cdt-II
fimH,fliC,stx1,etpD,katP
F
12
cdt-II
fimH,fliC,stx1,etpD,katP
F
13
cdt-II
fimH,fliC,stx1, etpD
B
14
cdt-III
fimH,fliC, espP,katP
D
15
cdt-III
fimH,fliC,hly,espP,katP
D
16
cdt-III
fimH,fliC,hly, etpD,katP
F
17
cdt-III
fimH,fliC,stx1, espP,sfpA
E
18
cdt-III
fimH,fliC,hly,stx1, katP
C
19
cdt-III
fimH,fliC,hly,stx1,stx2
M
20
cdt-III
fimH,fliC,hly,stx1, etpD
B
21
cdt-IV
fimH,fliC,stx1, espP,etpD,katP,sfpA
L
22
cdt-IV
fimH,fliC,hly,stx1, katP,sfpA
H
23
cdt-IV
fimH,fliC,stx1, etpD
B
24
cdt-IV
fimH,fliC,hly,stx1,stx2,espP,etpD,katP
K
25
cdt-IV
fimH,fliC,hly,stx1,etpD,sfpA
G
26
cdt-IV
fimH,fliC,stx1
M
27
cdt-IV
fimH,fliC,hly,stx1,katP
C
28
cdt-V
fimH,fliC,stx1,espP
A
29
cdt-V
fimH,fliC,hly,stx1, etpD,sfpA
G
30
cdt-V
fimH,fliC,hly,stx1, etpD,sfpA
GThe most prominent F profile with 20% frequency was repeated 6 times (etpD/katP). The absence of plasmid genes was shown in 10% of strains. Frequency of all the plasmid born associated genes with L profile was only 3.33% (espP/etpD/katP/sfpA).The higher prevalence of profile F (etpD/katP) is considerable (20%) and then profile C (katP) is more prevalent (16.68%). The frequency of profiles B (etpD), D (espP/katP), G (etpD/sfpA), and M (absence of plasmid-borne genes) was 10%. The frequency of other remaining profiles was 3.33% exceptionally and profile I (etpD/katP/sfpA) is 6.67%.Incdt-I group, 50% I profile (etpD/katP/sfpA) was shown, and in 40% of strains, C profile (katP) was demonstrated. Incdt-II group, 50% strains also showed I profile (etpD/katP/sfpA) and in 66.67% of strains F profile (etpD/katP) (the most prominent profile) was detected. The prominent profiles incdt-III group were 100% E profile (espP/sfpA) and 66.67% D profile (espP/katP). H, K, and L profiles are the most frequent profiles (100%) incdt-IV group. Incdt-V group, 100% of strains have A profile (espP) and 66.67% showed G profile (etpD/sfpA). In eachcdt group specific plasmid born profile was shown in Table 5.Table 5
The frequency of differentcdt-types based on virulent plasmid-borne genes in isolates totally.
Profile
Target gene
cdt-I
cdt-II
cdt-III
cdt-IV
cdt-V
A
0%
0%
0%
0%
100%
B
0%
33.33%
33.33%
33.33%
0%
C
40%
20%
20%
20%
0%
D
33.33%
0%
66.67%
0%
0%
E
0%
0%
100%
0%
0%
F
16.67%
66.67%
16.67%
0%
0%
G
0%
0%
0%
33.33%
66.67%
H
0%
0%
0%
100%
0%
I
50%
50%
0%
0%
0%
K
0%
0%
0%
100%
0%
L
0%
0%
0%
100%
0%
M
33.33%
0%
33.33%
33.33%
0%The most prominent gene in each group was determined.katP plasmid gene was presented 83.3% incdt-I and 85.7% incdt-II groups. TheetpD gene presentation was 85.7% incdt-II group and 66.66% incdt-IV group;espP presentation was 42.85% and 33.33% incdt-III andcdt-V group, respectively. ThesfpA presentation incdt-IV andcd-tV group was 42.85% and 66.66%, respectively.


### 3.2. Dendogram Clustering of Strains

In Figure1, hierarchical clustering analysis based on average linkage and rescaled distance cluster combine was done. The pattern of virulence gene association was compared and the linkage of these strains was illustrated. In this dendogram, association ofcdt-I,cdt-II (branch 4) and genetic linkage ofcdt-IV,cdt-V (branch 3) groups was shown in branch 1. The genotype association ofcdt-III was also shown in a completely separated branch (branch 2).Figure 1
Dendrogram using average linkage between groups: This figure illustrates hierarchical clustering analyses between 30 examined clinical isolates which are clustered based on virulence-associated factors including virulent plasmid, phage, chromosomal, and pathogenicity island genes. At the end of each branch the numbers indicate strains, as moving to the right relative distance becomes more apparent. Distances are more distinct by using a 0 to 25 scale along the top of the chart. Depicted table which is in front of the tree illustrates the comparative patterns of cooccurrence and presence of virulent genes in different strains (light gray scale shows gene loss and different dark gray scales show different virulent genes genesis). Defined pattern can be observed for eachcdt-type isolates based on other virulent genes.


## 3.1. Virulent Genes Detection

Existence of virulence genes, includingespP,etpD,katP,sfpA,hly,fliC,fimH,stx1,stx2, andcdt genes, was assessed by PCR (Table 2).Table 2
The occurrence of virulence-associated genes including plasmid, Pathogenicity Island, chromosomal and phage genes. (+) indicates positive PCR product and the existence of mentioned gene while (−) indicates negative PCR product.
Strain
espP
etpD
katP
sfpA
Hly
fliC
fimH
cdt-I
cdt-II
cdt-III
cdt-IV
cdt-V
stx1
stx2
1
−
+
+
+
−
+
+
+
−
−
−
−
−
−
2
−
−
−
−
−
−
+
+
−
−
−
−
+
−
3
−
−
+
−
−
+
+
+
−
−
−
−
+
−
4
+
−
+
−
−
+
+
+
−
−
−
−
+
−
5
−
−
+
−
−
+
+
+
−
−
−
−
+
+
6
−
+
+
−
−
+
+
+
−
−
−
−
+
+
7
−
+
+
+
−
+
+
−
+
−
−
−
+
−
8
−
+
+
−
−
+
+
−
+
−
−
−
+
−
9
−
−
+
−
+
+
+
−
+
−
−
−
+
−
10
−
+
+
−
−
+
+
−
+
−
−
−
+
−
11
−
+
+
−
−
+
+
−
+
−
−
−
+
−
12
−
+
+
−
−
+
+
−
+
−
−
−
+
−
13
−
+
−
−
−
+
+
−
+
−
−
−
+
−
14
+
−
+
−
−
+
+
−
−
+
−
−
−
−
15
+
−
+
−
+
+
+
−
−
+
−
−
−
−
16
−
+
+
−
+
+
+
−
−
+
−
−
−
−
17
+
−
−
+
−
+
+
−
−
+
−
−
+
−
18
−
−
+
−
+
+
+
−
−
+
−
−
+
−
19
−
−
−
−
+
+
+
−
−
+
−
−
+
+
20
−
+
−
−
+
+
+
−
−
+
−
−
+
−
21
+
+
+
+
−
+
+
−
−
−
+
−
+
−
22
−
−
+
+
+
+
+
−
−
−
+
−
+
−
23
−
+
−
−
−
+
+
−
−
−
+
−
+
−
24
+
+
+
−
+
+
+
−
−
−
+
−
+
+
25
−
+
−
+
+
+
+
−
−
−
+
−
+
−
26
−
−
−
−
−
+
+
−
−
−
+
−
+
−
27
−
−
+
−
+
+
+
−
−
−
+
−
+
−
28
+
−
−
−
−
+
+
−
−
−
−
+
+
−
29
−
+
−
+
+
+
+
−
−
−
−
+
+
−
30
−
+
−
+
+
+
+
−
−
−
−
+
+
−All strains werecdt positive and regardingcdt types 6 strains werecdt-I (20%), 7 strains (23.3%) werecdt-II,cdt-III, andcdt-IV, and 3 strains were recognized ascdt-V (10%).In the present study,stx1 gene was present in 86.7% of clinical isolates. However, thestx2 gene was detected in 13.3% of strains.stx1 gene was also detected in 83.3% ofcdt-type-I producing strains, while it was detected in all thecdt-typeII,IV,V strains and in 57.1% ofcdt-III isolates (Table 3).stx2 gene was present at 33.3% ofcdt-type I strains, 14.3% incdt-typeIII,IV strains, while in CDT-II and CDT-V producing isolates it was not observed.Table 3
The frequency of target genes in differentcdt-type isolates.
cdt type
Target gene
katP
etpD
espP
sfpA
stx1
stx2
hly
cdt-type I
83.33%
33.33%
16.6%
16.66%
83.33%
33.33%
0%
cdt-type II
85.7%
85.7%
0%
14.28%
100%
0%
14.28%
cdt-type III
57.14%
28.57%
42.85%
14.28%
57.14%
14.28%
71.42%
cdt-type IV
57.14%
57.4%
28.57%
42.85%
100%
14.28%
57.14%
cdt-type V
0%
66.66%
33.33%
66.66%
100%
0%
66.66%hly gene was not detected in any of CDT-I-producing strains. However, it was detected in 14.3% ofcdt-II strains, 71.4% ofcdt-III strains, 57.1% ofcdt-IV strains, and 66.7% of CDT-V-producing isolates (Table 3). In this clinical isolates, 53.3%etpD, 63.3%katP,and 23.3%espPgenes were detected. In the present study,etpD gene was observed in 33.3% ofcdt-type I, 85.7% ofcdt-type II, 28.6% ofcdt-type III, 57.1% ofcdt-type IV, and 66.7% ofcdt-type V strains.espP gene was present in 16.7% ofcdt-type I, 42.9% of CDT-III-producing, 28.6% ofcdt-type IV, 33.3% ofcdt-type V strains, while, in CDT-II-producing isolates, the gene was not detected. In addition,katP gene was detected in 83.3% ofcdt-type I, in 85.7% ofcdt-type II, in 57.1% of CDT-III- and CDT-IV-producing strains, while, this gene was not observed in CDT-V-producing strains (Table 3).Occurrence of virulent genes,espP,etpD,katP, andsfpA, was classified as an A–M pattern which was based on the existence of plasmid encoding genes (Table 4).Table 4
Differentcdt-type strains and related genotype based on other virulent genes and profile designation based on cooccurrence of plasmid-borne genes. espP: profile A, etpD: profile B, katP: profile C, espP/katP: profileD, espP/sfpA: profile E, etpD/katP: profile F, etpD/sfpA: profile G, katP/sfpA: profile H, etpD/katP/sfpA: profile I, espP/etpD/katP: profile K, espP/etpD/katP/sfpA: profile L, and  profile M: Indicates the absence of plasmid genes and presence of chromosomal genes (fimH, fliC, hly, stx1, or stx2).
Strain
cdt-type
Genotype based on virulence-related genes
Profile designation based on plasmid-borne genes
1
cdt-I
fimH,fliC, etpD,katP,sfpA
I
2
cdt-I
fimH,stx1
M
3
cdt-I
fimH,fliC,stx1, katP
C
4
cdt-I
fimH,fliC,stx1, espP,katP
D
5
cdt-I
fimH,fliC,stx1,stx2, katP
C
6
cdt-I
fimH,fliC,stx1,stx2, etpD,katP
F
7
cdt-II
fimH,fliC,stx1,etpD,katP,sfpA
I
8
cdt-II
fimH,fliC,stx1,etpD,katP
F
9
cdt-II
fimH,fliC,hly,stx1,katP
C
10
cdt-II
fimH,fliC,stx1,etpD,katP
F
11
cdt-II
fimH,fliC,stx1,etpD,katP
F
12
cdt-II
fimH,fliC,stx1,etpD,katP
F
13
cdt-II
fimH,fliC,stx1, etpD
B
14
cdt-III
fimH,fliC, espP,katP
D
15
cdt-III
fimH,fliC,hly,espP,katP
D
16
cdt-III
fimH,fliC,hly, etpD,katP
F
17
cdt-III
fimH,fliC,stx1, espP,sfpA
E
18
cdt-III
fimH,fliC,hly,stx1, katP
C
19
cdt-III
fimH,fliC,hly,stx1,stx2
M
20
cdt-III
fimH,fliC,hly,stx1, etpD
B
21
cdt-IV
fimH,fliC,stx1, espP,etpD,katP,sfpA
L
22
cdt-IV
fimH,fliC,hly,stx1, katP,sfpA
H
23
cdt-IV
fimH,fliC,stx1, etpD
B
24
cdt-IV
fimH,fliC,hly,stx1,stx2,espP,etpD,katP
K
25
cdt-IV
fimH,fliC,hly,stx1,etpD,sfpA
G
26
cdt-IV
fimH,fliC,stx1
M
27
cdt-IV
fimH,fliC,hly,stx1,katP
C
28
cdt-V
fimH,fliC,stx1,espP
A
29
cdt-V
fimH,fliC,hly,stx1, etpD,sfpA
G
30
cdt-V
fimH,fliC,hly,stx1, etpD,sfpA
GThe most prominent F profile with 20% frequency was repeated 6 times (etpD/katP). The absence of plasmid genes was shown in 10% of strains. Frequency of all the plasmid born associated genes with L profile was only 3.33% (espP/etpD/katP/sfpA).The higher prevalence of profile F (etpD/katP) is considerable (20%) and then profile C (katP) is more prevalent (16.68%). The frequency of profiles B (etpD), D (espP/katP), G (etpD/sfpA), and M (absence of plasmid-borne genes) was 10%. The frequency of other remaining profiles was 3.33% exceptionally and profile I (etpD/katP/sfpA) is 6.67%.Incdt-I group, 50% I profile (etpD/katP/sfpA) was shown, and in 40% of strains, C profile (katP) was demonstrated. Incdt-II group, 50% strains also showed I profile (etpD/katP/sfpA) and in 66.67% of strains F profile (etpD/katP) (the most prominent profile) was detected. The prominent profiles incdt-III group were 100% E profile (espP/sfpA) and 66.67% D profile (espP/katP). H, K, and L profiles are the most frequent profiles (100%) incdt-IV group. Incdt-V group, 100% of strains have A profile (espP) and 66.67% showed G profile (etpD/sfpA). In eachcdt group specific plasmid born profile was shown in Table 5.Table 5
The frequency of differentcdt-types based on virulent plasmid-borne genes in isolates totally.
Profile
Target gene
cdt-I
cdt-II
cdt-III
cdt-IV
cdt-V
A
0%
0%
0%
0%
100%
B
0%
33.33%
33.33%
33.33%
0%
C
40%
20%
20%
20%
0%
D
33.33%
0%
66.67%
0%
0%
E
0%
0%
100%
0%
0%
F
16.67%
66.67%
16.67%
0%
0%
G
0%
0%
0%
33.33%
66.67%
H
0%
0%
0%
100%
0%
I
50%
50%
0%
0%
0%
K
0%
0%
0%
100%
0%
L
0%
0%
0%
100%
0%
M
33.33%
0%
33.33%
33.33%
0%The most prominent gene in each group was determined.katP plasmid gene was presented 83.3% incdt-I and 85.7% incdt-II groups. TheetpD gene presentation was 85.7% incdt-II group and 66.66% incdt-IV group;espP presentation was 42.85% and 33.33% incdt-III andcdt-V group, respectively. ThesfpA presentation incdt-IV andcd-tV group was 42.85% and 66.66%, respectively.


## 3.2. Dendogram Clustering of Strains

In Figure1, hierarchical clustering analysis based on average linkage and rescaled distance cluster combine was done. The pattern of virulence gene association was compared and the linkage of these strains was illustrated. In this dendogram, association ofcdt-I,cdt-II (branch 4) and genetic linkage ofcdt-IV,cdt-V (branch 3) groups was shown in branch 1. The genotype association ofcdt-III was also shown in a completely separated branch (branch 2).Figure 1
Dendrogram using average linkage between groups: This figure illustrates hierarchical clustering analyses between 30 examined clinical isolates which are clustered based on virulence-associated factors including virulent plasmid, phage, chromosomal, and pathogenicity island genes. At the end of each branch the numbers indicate strains, as moving to the right relative distance becomes more apparent. Distances are more distinct by using a 0 to 25 scale along the top of the chart. Depicted table which is in front of the tree illustrates the comparative patterns of cooccurrence and presence of virulent genes in different strains (light gray scale shows gene loss and different dark gray scales show different virulent genes genesis). Defined pattern can be observed for eachcdt-type isolates based on other virulent genes.


## 4. Discussion

In the current study, we analyzed differentE. coli strains isolated from clinical diarrheal patients in order to obtain evidence of existence between virulence genotype and differentcdt types. It has been shown that CDT production has been associated with pathogenicE. coli. Both the low prevalence ofcdt genes and their association with other virulent genes suggest that thecdt genes are acquired independently in a number ofE. coli lineages, possibly as a result of horizontal gene transfer [7]. Five different CDTs have been reported forE. coli, so far [5]. It has also been shown thatcdt-I andcdt-IV genes appeared to belong to the same phylogenetic lineage, whereas thecdt-II,cdt-III, andcdt-V genes are clustered together in another lineage. This clustering profile was based on genomic extend diversity. In our study, regarding the assessed virulent genes two branches were first separated into branch 1 (includingcdt-I,cdt-II andcdt-IV,cdt-V genes) and branch 2,cdt-III genes. These data suggest thatcdt genes were acquired by horizontal transfer events at some point and evolved separately since then [5]. Our study shows complete separation ofcdt-III from other cdt types that agree with the finding that this group originated from animals and the other groups are more belonging to human. Furthermore,cdt-I,cdt-II (branch 4) andcdt-IV,cdt-V (branch 3) groups are more similar regarding virulence genes and originated from same progeny.It is now evident thathly gene is located on a PAI and encodes alpha-hemolysin.The production of hemolysin incdt-III-,cdt-IV-producing human and animal pathogenicE. coli strains was observed frequently [5]. Thus, not surprisingly,hly was detected in 40% of our clinical isolates. In concurrence with other studies, the prevalence ofhly gene incdt-type III, IV, V was more than other isolates. These results demonstrate that, possibly, there could be a relationship between the existence ofhly gene and the type ofcdt gene in clinicalE. coli isolates.fliC andfim are the two chromosomally located genes analyzed in our study.fliC gene was detected in 96.7% of our isolates.fliC gene encodes the flagellin subunit and could be considered as genomic background ofE. coli. Thus it could be expected to detectfliC in the most of our isolates. Similar to our findings, all the isolates havefliC gene in an experiment performed in Finland in 2006 [23]. Type 1 fimbriae are also encoded by the chromosomally locatedfim gene cluster. The presence offim DNA sequences is common amongE. coli strains. In fact, the majority of clinical isolates, both virulent and avirulent, could be induced to express type 1 fimbriae.Among pathogenicE. coli, the existence of a large virulent plasmid (pO157) has been observed. TheetpD,katP, andespP genes are located on this plasmid. pO157 plasmid is mainly associated with EHEC and ETEC strains. In our study, 53.3% of clinical isolates harboretpD, 63.3%katP and 23.3%espP genes. There is a relation between the occurrence ofstx genes and these virulent plasmid-associated genes [24]. Moreover, PCR analysis revealed a close relationship between the occurrence of plasmid-bornekatP gene andstx gene in pathogenicE. coli [12]. Most ofkatP+ strains belong to shiga toxin-producingE. coli [24]. Our results were similar to the findings by Beutin et al., 2005 [24, 27]. Based on our findings, it could be deduced thatkatP gene is mostly present in CDT-I- and CDT-II-producing strains.ESpP, which possesses human coagulation factor V and pepsin A proteolytic activity, is the significant marker of virulence in shiga toxin-producing strains [28]. High frequency ofespP in CDT-III-producing isolates is considerable.Alpha-hemolysin is frequently associated with human uropathogenicE. coli (UPEC); furthermore related encoding PAI is also so instable and the operon could be located on either a plasmid or the chromosome [22, 29]. Besides urinary tract infection (UTI) is caused predominantly by type 1-fimbriated UPEC and initial binding is mediated by the FimH adhesin of the mentioned fimbriae [30]. In our study in 100% ofhly
+ strains,fimH gene was detected. In addition, allhly
+ strains possess one or more of plasmid pO157 genes includingetpD,katP, andespP. These genes plusstx gene are one of the EHEC and STEC characteristics althoughespP gene is common in EPEC and EHEC [14, 31]. Simultaneous presence of these genes indicates that our isolates obtainhly operon and relevant PAI through the horizontal gene transfer. In addition, in evolutionary pathway, isolates by achieving thecdt genes improve their pathogenicity.Our findings demonstrate that considering virulence genes CDT-producing strains belong to the heterogeneous group. The branching-type nature of dendrogram allows us to cluster the strains at various levels. Moreover, it gives an idea of how great the distance is between the cases that are clustered in a particular step, using a 0 to 25 scale along the top of the chart (Figure1). Strains which are clustered as a particular group or close together in dendrogram are likely to have similar characteristics while possess their own unique genotype and genomic content. In the same cluster, one can also observe a relatively unique pattern of virulent genes which is shown in Figure 1. For instance, each distinctcdt-type group, by possessing a particularcdt gene as genomic backbone, has an approximately similar pattern based on other virulent genes. For example, profile F (etpD/katP) is more prevalent incdt-type II group orhly gene is prevalent incdt-type III isolates.In addition, this phenomenon is also observed in each group belonging to a particular plasmid-profile, so thatcdt-V gene is the most prevalent in profile G (etpD/sfpA). This evidence further confirms that horizontal gene transfer could occur among pathogenic strains.Association ofcdt-I,cdt-II andcdt-IV,cdt-V genotype groups is depicted in the dendogram. Accordingly,cdt-III genotype association has been shown in a separated branch. On the other hand, from 3 strains ofcdt-V, two strains were associated withcdt-IV while one strain was associated withcdt-III.These findings may indicate that CDT-producing strains may have originated from a common ancestor but during their evolution by horizontal gene transfer, and they departed from each other.



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*Source: 9237127-2016-03-03.xml*