# Molecular Screening of Virulence Genes in Extraintestinal PathogenicEscherichia coli Isolated from Human Blood Culture in Brazil

Authors: Vanessa L. Koga; Geizecler Tomazetto; Paula S. Cyoia; Meiriele S. Neves; Marilda C. Vidotto; Gerson Nakazato; Renata K. T. Kobayashi
Journal: BioMed Research International
 (2014)

---

## Abstract

Extraintestinal pathogenicEscherichia coli (ExPEC) is one of the main etiological agents of bloodstream infections caused by Gram-negative bacilli. In the present study, 20 E. coli isolates from human hemocultures were characterized to identify genetic features associated with virulence (pathogenicity islands markers, phylogenetic group, virulence genes, plasmid profiles, and conjugative plasmids) and these results were compared with commensal isolates. The most prevalent pathogenicity island, in strains from hemoculture, were PAI IV536, described by many researchers as a stable island in enterobacteria. Among virulence genes, iutA gene was found more frequently and this gene enconding the aerobactin siderophore receptor. According to the phylogenetic classification, group B2 was the most commonly found. Additionally, through plasmid analysis, 14 isolates showed plasmids and 3 of these were shown to be conjugative. Although in stool samples of healthy people the presence of commensal strains is common, human intestinal tract may serve as a reservoir for ExPEC.


---

## Body

## 1. Introduction

Escherichia coliis one of the most common microorganisms of the human intestinal microbiota. However, a small percentage ofE. coli is capable of causing extraintestinal infections (extraintestinal pathogenicEscherichia coli—ExPEC), and these ExPECs are considered some of the main etiological agents of bacteremia caused by Gram-negative bacilli [1, 2]. According to the phylogenetic classification, ExPECs typically belong to group B2 and less commonly to group D, whereas commensal intestinal strains belong to group A or B1 [3]. ExPEC pathogenicity is due to the presence of genes, located on plasmids or chromosomes that encode virulence factors. When present on the chromosome, these genes are typically found in specific regions called pathogenicity islands (PAI). Given that the severity of bacterial infections is often due to the genetic features of the pathogenic agent and that few studies have investigated genetic aspects of ExPEC isolates from bacteremia in Brazil [4, 5], the aim of the present study was to characterize 20E. coli isolates from human hemocultures for genetic features associated with ExPEC. The investigation was based on the screening for PAI associated sequences, determination of phylogenetic group, genotypic identification of the major virulence factors of ExPEC, and plasmid analyses, and these results compared with commensal strains. This information may help us better understand the pathogenesis of these bacteria.


## 2. Materials and Methods

### 2.1. Bacterial Isolates

To perform the study, 20E. coli strains isolated from human hemocultures were kindly provided by Professor Marilda C. Vidotto (Brazil) [6] and 51E. coli strains were obtained from stools of 19 healthy Brazilians. The strains were stored in brain heart infusion with 20% glycerol at −20°C. The polymerase chain reaction (PCR) was used for the genetic characterization of PAI associated sequences, presence of virulence genes, and for phylogenetic classification. PCR amplicons were visualized on 1.0% agarose gels stained with GelRed (Biotium). After gel electrophoresis the images were captured using Image Capture Systems (LPixImageHE).


### 2.2. Detection of PAI Markers

The presence of sequences associated with seven different PAIs, previously characterized in uropathogenicE. coli (UPEC), was determined (PAI I536, II536, IV536, ICFT073, IICFT073, IJ96 and IIJ96) [7] (Table 1).Table 1
Primers for detection of PAIs markers, phylogenetic analysis, virulence genes, and their respective virulence factors.
Genes
Sequence (5′ to 3′)
Size of product (bp)
Virulence factors
Reference
PAI I
536
TAA TGC CGG AGA TTC ATT GTCAGG ATT TGT CTC AGG GCT TT
1.800
α-Haemolysin, CS12 fimbriae, and F17-like fimbrial adhesin
Sabaté et al., 2006 [7]
PAI II
536
CAT GTC CAA AGC TCG AGC CCTA CGT CAG GCT GGC TTT G
1.000
α-Haemolysin and P-related fimbriae
Sabaté et al., 2006 [7]
PAI IV
536
AAG GAT TCG CTG TTA CCG GACTCG TCG GGC AGC GTT TCT TCT
300
Yersiniabactin siderophore system
Sabaté et al., 2006 [7]
PAI I
CFT073
GGA CAT CCT GTT ACA GCG CGC ATCG CCA CCA ATC ACA GC GAA C
930
α-Haemolysin, P-fimbriae, and aerobactin
Sabaté et al., 2006 [7]
PAI II
CFT073
ATG GAT GTT GTA TCG CGC ACG AGC ATG TGG ATC TGC
400
P-fimbriae and iron-regulated genes
Sabaté et al., 2006 [7]
PAI I
J96
TCG TGC TCA GGT CCG GAA TTTTGG CAT CCC ACA TTA TCG
400
α-Haemolysin and P-fimbriae
Sabaté et al., 2006 [7]
PAI II
J96
GGA TCC ATG AAA ACA TGG TTA ATG GGGAT ATT TTT GTT GCC ATT GGT TAC C
2.300
α-Haemolysin, Prs-fimbriae, and cytotoxic necrotizing factor 1
Sabaté et al., 2006 [7]
chuA
GAC GAA CCA ACG GTC AGG ATTGC CGC CAG TAC CAA AGA CA
279
Hemetransport in enterohemorrhagic O157:H7E. coli
Clermont et al., 2000 [3]
yjaA
TGA AGT GTC AGG AGA CGC TGATG GAG AAT GCG TTC CTC AAC
211
Protein of function unknown
Clermont et al., 2000 [3]
TSPE4.C2
GAG TAA TGT CGG GGC ATT CACGC GCC AAC AAA GTA TTA CG
152
Putative DNA fragment (TSPE4.C2) inE. coli
Clermont et al., 2000 [3]
kpsII
GCG CAT TTG CTG ATA CTG TTGCAT CCA GAC GAT AAG CAT GAG CA
272
Capsule synthesis K1 e K5
Johnson and Stell, 2000 [8]
K1
TAG CAA ACG TTC TAT TGG TGCCAT CCA GAC GAT AAG CAT GAG CA
156
Capsule K1
Johnson and Stell, 2000 [8]
cvaC
CAC ACA CAA ACG GGA GCT GTTCTT CCC GCA GCA TAG TTC CAT
680
Colicin V
Johnson and Stell, 2000 [8]
iutA
GGC TGG ACA TCA TGG GAA CTG GCGT CGG GAA CGG GTA GAA TCG
302
Aerobactin siderophore receptor
Johnson et al., 2008 [9]
fyuA
TGA TTA ACC CCG CGA CGG AACGC AGT AGG CAC GAT CTT GTA
880
Yersiniabactin
Johnson and Stell, 2000 [8]
papC
GAC GGC TGT ACT GCA GGG TGT GGC GATA TCC TTT CTG CAG GCA GGG TGT GGC
328
P fimbriae
Le Bouguénec et al., 1992 [10]
papG
CTG TAA TTA CGG AAG TGA TTT CTGACT ATC CGG CTC CGG ATA AAC CAT
1070
P fimbriae
Johnson and Stell, 2000 [8]
sfaA
CTC CGG AGA ACT GGG TGC ATC TTA CCGG AGG AGT AAT TAC AAA CCT GGC A
410
Sfa fimbriae
Le Bouguénec et al., 1992 [10]
sfaS
GTG GAT ACG ACG ATT ACT GTGCCG CCA GCA TTC CCT GTA TTC
240
Sfa fimbriae
Johnson and Stell, 2000 [8]
Afa
GGC AGA GGG CCG GCA ACA GGCCCC GTA ACG CGC CAG CAT CTC
559
M fimbriae
Johnson and Stell, 2000 [8]
ibe10
AGG CAG GTG TGC GCC GCG TACTGG TGC TCC GGC AAA CCA TGC
170
Invasion of brain endothelium
Johnson and Stell, 2000 [8]
hlyA
AAC AAG GAT AAG CAC TGT TCT GGCACC ATA TAA GCG GTC ATT CCC GTC
1.177
Hemolysin
Johnson and Stell, 2000 [8]
cnf1
AGG ATG GAG TTT CCT ATG CAG GAGCAT TCA GAG TCC TGC CCT CAT TAT T
498
Cytotoxic necrotizing factor 1
Johnson and Stell, 2000 [8]
cnf2
AAT CTA ATT AAA GAG AACCAT GCT TTG TAT ATC TA
543
Cytotoxic necrotizing factor 2
Blanco et al., 1996 [34]
traT
GGT GTG GTG CGA TGA GCA CAGCAC GGT TCA GCC ATC CCT GAG
290
Serum resistance
Johnson and Stell, 2000 [8]
iroN
AAT CCG GCA AAG AGA CGA ACC GCC TGTT CGG GCA ACC CCT GCT TTG ACT TT
553
Salmochelin siderophore receptor
Johnson et al., 2008 [9]
ompT
TCA TCC CGG AAG CCT CCC TCA CTA CTA TTAG CGT TTG CTG CAC TGG CTT CTG ATA C
496
Episomal outer membrane protease
Johnson et al., 2008 [9]
hlyF
GGC CAC AGT CGT TTA GGG TGC TTA CCGGC GGT TTA GGC ATT CCG ATA CTC AG
450
Putative avian hemolysin
Johnson et al., 2008 [9]
Iss
CAG CAA CCC GAA CCA CTT GAT GAGC ATT GCC AGA GCG GCA GAA
323
Episomal increased serum survival
Johnson et al., 2008 [9]This PCR contained 1 U Taq DNA polymerase (Invitrogen) in 2x PCR buffer (Invitrogen), 0.2 mM of each dNTP, 2.5 mM MgCl2, and 20 pmol/μL of each primer (Table 1). The program consisted of 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension step at 72°C for 10 min [7]. The positive control used in the PCR was J96.


### 2.3. Phylogenetic Classification

Phylogenetic classification showed that theE. coli strains belonged to four groups (A, B1, B2, or D) based on the presence of thechuA andyjaA genes and the DNA fragment (TSPE4.C2). This PCR contained 1.25 U Taq DNA polymerase (Invitrogen) in 1x PCR buffer (Invitrogen), 20 pmol of each dNTP, 2.5 mM MgCl2, and 1 μM of each primer (Table 1). The program of PCR consisted of 94°C for 4 min, followed by 30 cycles of 94°C for 5 seg and 54°C for 10 seg, with a final extension step at 72°C for 5 min [3].


### 2.4. Virulence Factors Genes

The pathogenicity ofE. coliis associated with the presence of virulence factors that can be encoded by chromosomal and plasmid genes, and thus 19 genes encoding virulence factors were investigated. The genes selected were specific for hemolysins (hlyA andhlyF), cytotoxic necrotizing factors (cnf1 andcnf2), colicin V (cvaC), aerobactin (iutA), yersiniabactin (fyuA), salmochelin (iroN), P-fimbriae (papC andpapG), S-fimbrial adhesin (sfaA andsfaS), afimbrial adhesin (afa), serum resistance (issandtraT), brain microvascular endothelium invasion (ibe10), K1 capsule (kpsII and K1), and ompT outer membrane protein (ompT) [8–10]. This PCR contained 1.25 U Taq DNA polymerase (Invitrogen) in 1x PCR buffer (Invitrogen), 0.2 mM of each dNTP, 2.5 mM MgCl2, and 1 μM of each primer (Table 1). The program of PCR consisted of 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension step at 72°C for 10 min.


### 2.5. Plasmids Profile

To analyze the plasmid profile, the plasmids of wild strains and the plasmids R27 (110 MDa), JPN11 (66 MDa), PSA (23 MD), and pRK (13, 2 MDa) used as markers of molecular mass were extracted by alkaline lysis [11]; the molecular weight of the plasmids was measured (LabImage 1D software) and the ability to transfer was determined.The strains that harbored plasmids were chosen for mating experiments. The strains were grown in LB (Luria Bertani Broth) until the exponential phase. 1.2 mL of this culture was transferred to a flask containing 0.4 mL of the recipient culture in the stationary phase,E. coli K12-711 [12]. The mixture was incubated at 37°C for 3 hours. Transconjugants resistant to drugs were selected on MacConkey agar containing inhibitory concentrations of nalidixic acid (resistance present inE. coli K12-711), tetracycline, ampicillin, or kanamycin. The colonies grown on each selective plate were tested for the presence of virulence genes and pathogenicity islands, according to the virulence pattern of the donor strain. The resistance profile, necessary for this test, is shown in Table 2.Table 2
Plasmid and resistance profile of strains from hemocultures.
Strains
Plasmids
Molecular size of the plasmids (MDa)
Resistance profile
1
p1a
78
ApCbCfCmKnTr
2
p2a, b, c, d
113, 112, 86, 82
ApCbSmFoKnTr
3
p3a, b, c, d, e
112, 99, 78, 58, 29
ApCbCfCmSmFoGnKnSiSuTbTcTr
4
p4a
59
ApCfFoTcTr
5
p5a, b
99, 71
ApCbCfCmSmFoKnSuTcTr
6
p6a
112
ApCbCfCmSmFoGnKnSiTcTr
7
p7a
99
ApCbSmFoSu
8
NP
—
ApFo
9
NP
—
ApFo
10
p10a
82
ApCmFoTc
11
p11a
68
ApCbCmSmFo
12
NP
—
Ap
13
p13a, b, c, d, e
87, 68, 59, 50, 29
ApCbCfSmFoGnKnSuTbTcTr
14
p14a, b
62, 44
ApCbSm
15
NP
—
Ap
16
NP
—
Ap
17
p17a
87
ApCfCmSmKnTcTr
18
p18a
62
Ap
19
p19a
92
ApCbCmSmKnSuTcTr
20
NP
—
Ap
NP: no plasmid; Ap: ampicillin; Cb: carbenicillin; Cf: cephalothin; Cm: chloramphenicol; Sm: streptomycin; Fo: fosfomycin; Gn: gentamicin; Kn: kanamycin; Si: sisomicin; Su: sulfonamide; Tb: tobramycin; Tc: tetracycline; Tr: trimethoprim.


## 2.1. Bacterial Isolates

To perform the study, 20E. coli strains isolated from human hemocultures were kindly provided by Professor Marilda C. Vidotto (Brazil) [6] and 51E. coli strains were obtained from stools of 19 healthy Brazilians. The strains were stored in brain heart infusion with 20% glycerol at −20°C. The polymerase chain reaction (PCR) was used for the genetic characterization of PAI associated sequences, presence of virulence genes, and for phylogenetic classification. PCR amplicons were visualized on 1.0% agarose gels stained with GelRed (Biotium). After gel electrophoresis the images were captured using Image Capture Systems (LPixImageHE).


## 2.2. Detection of PAI Markers

The presence of sequences associated with seven different PAIs, previously characterized in uropathogenicE. coli (UPEC), was determined (PAI I536, II536, IV536, ICFT073, IICFT073, IJ96 and IIJ96) [7] (Table 1).Table 1
Primers for detection of PAIs markers, phylogenetic analysis, virulence genes, and their respective virulence factors.
Genes
Sequence (5′ to 3′)
Size of product (bp)
Virulence factors
Reference
PAI I
536
TAA TGC CGG AGA TTC ATT GTCAGG ATT TGT CTC AGG GCT TT
1.800
α-Haemolysin, CS12 fimbriae, and F17-like fimbrial adhesin
Sabaté et al., 2006 [7]
PAI II
536
CAT GTC CAA AGC TCG AGC CCTA CGT CAG GCT GGC TTT G
1.000
α-Haemolysin and P-related fimbriae
Sabaté et al., 2006 [7]
PAI IV
536
AAG GAT TCG CTG TTA CCG GACTCG TCG GGC AGC GTT TCT TCT
300
Yersiniabactin siderophore system
Sabaté et al., 2006 [7]
PAI I
CFT073
GGA CAT CCT GTT ACA GCG CGC ATCG CCA CCA ATC ACA GC GAA C
930
α-Haemolysin, P-fimbriae, and aerobactin
Sabaté et al., 2006 [7]
PAI II
CFT073
ATG GAT GTT GTA TCG CGC ACG AGC ATG TGG ATC TGC
400
P-fimbriae and iron-regulated genes
Sabaté et al., 2006 [7]
PAI I
J96
TCG TGC TCA GGT CCG GAA TTTTGG CAT CCC ACA TTA TCG
400
α-Haemolysin and P-fimbriae
Sabaté et al., 2006 [7]
PAI II
J96
GGA TCC ATG AAA ACA TGG TTA ATG GGGAT ATT TTT GTT GCC ATT GGT TAC C
2.300
α-Haemolysin, Prs-fimbriae, and cytotoxic necrotizing factor 1
Sabaté et al., 2006 [7]
chuA
GAC GAA CCA ACG GTC AGG ATTGC CGC CAG TAC CAA AGA CA
279
Hemetransport in enterohemorrhagic O157:H7E. coli
Clermont et al., 2000 [3]
yjaA
TGA AGT GTC AGG AGA CGC TGATG GAG AAT GCG TTC CTC AAC
211
Protein of function unknown
Clermont et al., 2000 [3]
TSPE4.C2
GAG TAA TGT CGG GGC ATT CACGC GCC AAC AAA GTA TTA CG
152
Putative DNA fragment (TSPE4.C2) inE. coli
Clermont et al., 2000 [3]
kpsII
GCG CAT TTG CTG ATA CTG TTGCAT CCA GAC GAT AAG CAT GAG CA
272
Capsule synthesis K1 e K5
Johnson and Stell, 2000 [8]
K1
TAG CAA ACG TTC TAT TGG TGCCAT CCA GAC GAT AAG CAT GAG CA
156
Capsule K1
Johnson and Stell, 2000 [8]
cvaC
CAC ACA CAA ACG GGA GCT GTTCTT CCC GCA GCA TAG TTC CAT
680
Colicin V
Johnson and Stell, 2000 [8]
iutA
GGC TGG ACA TCA TGG GAA CTG GCGT CGG GAA CGG GTA GAA TCG
302
Aerobactin siderophore receptor
Johnson et al., 2008 [9]
fyuA
TGA TTA ACC CCG CGA CGG AACGC AGT AGG CAC GAT CTT GTA
880
Yersiniabactin
Johnson and Stell, 2000 [8]
papC
GAC GGC TGT ACT GCA GGG TGT GGC GATA TCC TTT CTG CAG GCA GGG TGT GGC
328
P fimbriae
Le Bouguénec et al., 1992 [10]
papG
CTG TAA TTA CGG AAG TGA TTT CTGACT ATC CGG CTC CGG ATA AAC CAT
1070
P fimbriae
Johnson and Stell, 2000 [8]
sfaA
CTC CGG AGA ACT GGG TGC ATC TTA CCGG AGG AGT AAT TAC AAA CCT GGC A
410
Sfa fimbriae
Le Bouguénec et al., 1992 [10]
sfaS
GTG GAT ACG ACG ATT ACT GTGCCG CCA GCA TTC CCT GTA TTC
240
Sfa fimbriae
Johnson and Stell, 2000 [8]
Afa
GGC AGA GGG CCG GCA ACA GGCCCC GTA ACG CGC CAG CAT CTC
559
M fimbriae
Johnson and Stell, 2000 [8]
ibe10
AGG CAG GTG TGC GCC GCG TACTGG TGC TCC GGC AAA CCA TGC
170
Invasion of brain endothelium
Johnson and Stell, 2000 [8]
hlyA
AAC AAG GAT AAG CAC TGT TCT GGCACC ATA TAA GCG GTC ATT CCC GTC
1.177
Hemolysin
Johnson and Stell, 2000 [8]
cnf1
AGG ATG GAG TTT CCT ATG CAG GAGCAT TCA GAG TCC TGC CCT CAT TAT T
498
Cytotoxic necrotizing factor 1
Johnson and Stell, 2000 [8]
cnf2
AAT CTA ATT AAA GAG AACCAT GCT TTG TAT ATC TA
543
Cytotoxic necrotizing factor 2
Blanco et al., 1996 [34]
traT
GGT GTG GTG CGA TGA GCA CAGCAC GGT TCA GCC ATC CCT GAG
290
Serum resistance
Johnson and Stell, 2000 [8]
iroN
AAT CCG GCA AAG AGA CGA ACC GCC TGTT CGG GCA ACC CCT GCT TTG ACT TT
553
Salmochelin siderophore receptor
Johnson et al., 2008 [9]
ompT
TCA TCC CGG AAG CCT CCC TCA CTA CTA TTAG CGT TTG CTG CAC TGG CTT CTG ATA C
496
Episomal outer membrane protease
Johnson et al., 2008 [9]
hlyF
GGC CAC AGT CGT TTA GGG TGC TTA CCGGC GGT TTA GGC ATT CCG ATA CTC AG
450
Putative avian hemolysin
Johnson et al., 2008 [9]
Iss
CAG CAA CCC GAA CCA CTT GAT GAGC ATT GCC AGA GCG GCA GAA
323
Episomal increased serum survival
Johnson et al., 2008 [9]This PCR contained 1 U Taq DNA polymerase (Invitrogen) in 2x PCR buffer (Invitrogen), 0.2 mM of each dNTP, 2.5 mM MgCl2, and 20 pmol/μL of each primer (Table 1). The program consisted of 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension step at 72°C for 10 min [7]. The positive control used in the PCR was J96.


## 2.3. Phylogenetic Classification

Phylogenetic classification showed that theE. coli strains belonged to four groups (A, B1, B2, or D) based on the presence of thechuA andyjaA genes and the DNA fragment (TSPE4.C2). This PCR contained 1.25 U Taq DNA polymerase (Invitrogen) in 1x PCR buffer (Invitrogen), 20 pmol of each dNTP, 2.5 mM MgCl2, and 1 μM of each primer (Table 1). The program of PCR consisted of 94°C for 4 min, followed by 30 cycles of 94°C for 5 seg and 54°C for 10 seg, with a final extension step at 72°C for 5 min [3].


## 2.4. Virulence Factors Genes

The pathogenicity ofE. coliis associated with the presence of virulence factors that can be encoded by chromosomal and plasmid genes, and thus 19 genes encoding virulence factors were investigated. The genes selected were specific for hemolysins (hlyA andhlyF), cytotoxic necrotizing factors (cnf1 andcnf2), colicin V (cvaC), aerobactin (iutA), yersiniabactin (fyuA), salmochelin (iroN), P-fimbriae (papC andpapG), S-fimbrial adhesin (sfaA andsfaS), afimbrial adhesin (afa), serum resistance (issandtraT), brain microvascular endothelium invasion (ibe10), K1 capsule (kpsII and K1), and ompT outer membrane protein (ompT) [8–10]. This PCR contained 1.25 U Taq DNA polymerase (Invitrogen) in 1x PCR buffer (Invitrogen), 0.2 mM of each dNTP, 2.5 mM MgCl2, and 1 μM of each primer (Table 1). The program of PCR consisted of 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension step at 72°C for 10 min.


## 2.5. Plasmids Profile

To analyze the plasmid profile, the plasmids of wild strains and the plasmids R27 (110 MDa), JPN11 (66 MDa), PSA (23 MD), and pRK (13, 2 MDa) used as markers of molecular mass were extracted by alkaline lysis [11]; the molecular weight of the plasmids was measured (LabImage 1D software) and the ability to transfer was determined.The strains that harbored plasmids were chosen for mating experiments. The strains were grown in LB (Luria Bertani Broth) until the exponential phase. 1.2 mL of this culture was transferred to a flask containing 0.4 mL of the recipient culture in the stationary phase,E. coli K12-711 [12]. The mixture was incubated at 37°C for 3 hours. Transconjugants resistant to drugs were selected on MacConkey agar containing inhibitory concentrations of nalidixic acid (resistance present inE. coli K12-711), tetracycline, ampicillin, or kanamycin. The colonies grown on each selective plate were tested for the presence of virulence genes and pathogenicity islands, according to the virulence pattern of the donor strain. The resistance profile, necessary for this test, is shown in Table 2.Table 2
Plasmid and resistance profile of strains from hemocultures.
Strains
Plasmids
Molecular size of the plasmids (MDa)
Resistance profile
1
p1a
78
ApCbCfCmKnTr
2
p2a, b, c, d
113, 112, 86, 82
ApCbSmFoKnTr
3
p3a, b, c, d, e
112, 99, 78, 58, 29
ApCbCfCmSmFoGnKnSiSuTbTcTr
4
p4a
59
ApCfFoTcTr
5
p5a, b
99, 71
ApCbCfCmSmFoKnSuTcTr
6
p6a
112
ApCbCfCmSmFoGnKnSiTcTr
7
p7a
99
ApCbSmFoSu
8
NP
—
ApFo
9
NP
—
ApFo
10
p10a
82
ApCmFoTc
11
p11a
68
ApCbCmSmFo
12
NP
—
Ap
13
p13a, b, c, d, e
87, 68, 59, 50, 29
ApCbCfSmFoGnKnSuTbTcTr
14
p14a, b
62, 44
ApCbSm
15
NP
—
Ap
16
NP
—
Ap
17
p17a
87
ApCfCmSmKnTcTr
18
p18a
62
Ap
19
p19a
92
ApCbCmSmKnSuTcTr
20
NP
—
Ap
NP: no plasmid; Ap: ampicillin; Cb: carbenicillin; Cf: cephalothin; Cm: chloramphenicol; Sm: streptomycin; Fo: fosfomycin; Gn: gentamicin; Kn: kanamycin; Si: sisomicin; Su: sulfonamide; Tb: tobramycin; Tc: tetracycline; Tr: trimethoprim.


## 3. Results and Discussion

Extraintestinal pathogenicEscherichia coli(ExPEC) is one of the leading causes of bloodstream infections (BSI) worldwide. In Brazil, it was observed a high mortality associated with BSI [13]. Despite the importance ofE. coli bloodstream infections due to their high morbidity and mortality, the pathogenesis is not well known [14, 15] and not studied enough in South America [4, 5].In this study we screened 20 strains from bacteremia from newborns (10%), children aged between 6 months and 14 years (20%), and adults (70%) and from two Brazilian teaching hospitals for the determination of phylogenetic group, pathogenicity islands (PAI) associated sequences, and important virulence factors responsible for extraintestinal pathogenesis. The results were compared with those obtained with isolates from the stools of healthy humans. The relationship between the presence of PAIs, virulence genes, and the phylogenetic group was analyzed.Among the 20 isolates from the hemocultures tested, 70% of the isolates displayed PAI associated sequences (total of 22 islands), while in the commensal strains 52.94% of them displayed PAI (total of 45 islands). In agreement with previously published data [7, 16, 17], PAI IV536 was the most prevalent in both groups, following the PAI ICFT073 and PAI IICFT073, as shown in Tables 3 and 4. PAI IV536 has been described by many researchers as a stable island, and it is one of the most commonly found PAIs in enterobacteria [7, 18]. Our results show that the islands present in UPEC, although poorly researched in septicemic strains, are also found inE. coli isolated from hemocultures. This similarity can be associated with the fact that the urinary tract infection is one of the most common infections and bacteraemia is often a complication of this infection. But there are other ways for the presence of bacteria in the blood, such as meningitis and polymicrobial intra-abdominal infections. The presence of bacteria in the bloodstream suggests the ability of these pathogens to survive in an environment with scarce free iron and to resist the bactericidal activity in the blood [4].Table 3
Study of pathogenicity islands, phylogenetic classification, and virulence genes in 20 ExPEC strains from human hemocultures.
Strains
PC
Pathogenicity islands
Virulence genes
IV536
ICFT073
IICFT073
IJ96
kpsII
K1
fyuA
papC
papG
sfaA
Ibe10
hly
cnf1
cnf2
traT
iutA
iroN
ompT
hlyF
iss
1
B2
+
+
+
−
+
−
−
+
+
+
−
+
−
−
−
+
+
−
−
−
2
B2
+
+
+
−
+
+
−
−
−
−
−
−
−
−
−
+
+
−
−
−
3
B2
+
+
−
−
+
+
−
−
−
−
−
−
−
−
−
+
−
−
−
−
4
B2
−
−
−
+
+
+
−
−
−
−
+
−
−
−
−
+
−
−
−
−
5
A
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
−
+
+
+
+
6
A
−
−
−
−
−
−
−
+
+
−
−
−
−
−
+
+
+
−
−
−
7
B2
+
+
−
−
+
−
+
+
+
−
−
+
−
−
+
+
+
−
−
−
8
B2
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
9
B2
+
+
−
−
+
+
+
+
+
+
−
+
−
−
−
+
−
−
−
−
10
B1
+
−
−
−
−
−
+
−
−
−
−
+
−
+
+
−
+
+
+
+
11
A
+
−
−
−
−
−
+
−
−
−
−
+
−
−
+
+
+
−
−
−
12
B1
+
−
−
−
−
−
+
−
−
−
+
−
−
−
−
−
−
−
−
−
13
A
−
−
−
−
+
+
+
+
+
−
−
+
−
−
+
+
−
−
−
−
14
A
−
−
−
−
+
+
−
+
+
−
−
−
−
−
+
+
+
−
−
−
15
A
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
−
−
−
−
16
B2
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
−
−
−
17
D
+
−
−
−
−
−
+
−
−
−
−
−
−
−
+
+
−
−
−
−
18
B1
+
−
−
−
−
−
+
−
−
−
−
−
−
−
+
−
−
−
−
−
19
D
+
−
−
−
−
−
+
−
−
−
−
−
−
−
+
+
+
−
−
+
20
B1
−
−
+
−
−
−
−
−
−
−
−
−
−
−
−
−
+
+
−
+
PC: phylogenetic classification; pathogenicity islands: PAI IV536, PAI ICFT073, PAI IICFT073, and PAI IJ96; adhesions: papC, papG, sfaA, sfaS, and afa; invasion: ibe10; toxins: cnf2, cnf1, hlyA, hlyF, and cvaC; iron uptake systems: fyuA, iutA, and iroN; serum resistance: kps II, K1, traT, and iss; proteases: ompT.Table 4
Distribution of pathogenicity islands (PAI) according to phylogenetic classification (PC), among ExPEC and commensal strains.
PC
PAI I536
PAI II536
PAI IV536
PAI IJ96
PAI ICFT073
PAI IICFT073
Total of PAIs
Total of strains
B2 (ExPEC)
0
0
6
1
6
2
15
8
B2 (commensal)
0
0
2
0
2
1
5
6
D (ExPEC)
0
0
2
0
0
0
2
2
D (commensal)
0
0
0
0
0
0
0
3
B1 (ExPEC)
0
0
3
0
0
1
3
4
B1 (commensal)
0
0
0
0
2
0
2
5
A (ExPEC)
0
0
1
0
0
0
1
6
A (commensal)
3
3
21
0
7
4
38
37It is not clear yet if allE. coli from the intestinal tract of healthy people can be considered commensal, as some isolates showed up to five pathogenicity islands. Already it has been reported that ExPEC can asymptomatically colonize the intestinal tract [7].In this study, genes related to toxin and hemolysin production were researched and includedcnf2,cnf1,hlyA,hlyF, andcvaC. ThehlyA gene is frequently detected in ExPEC and it was the most prevalent gene in our strains of hemoculture (30%) [19, 20].Escherichia colihemolysin (hlyA) is a pore-forming bacterial exotoxin that may contribute to the virulence of bacteria during bloodstream infection and sepsis [20, 21]. The PAIs I536, II536, ICFT073, IJ96, and IIJ96 harbor a copy of hlyABCD system encoding α-hemolysin, and in our study, six strains harborhlyA gene and three also had these PAIs (Table 3). Commensal strains showed a large prevalence of thehlyA gene too, with 52.94% of the strains, and did not show a good correlation with the PAIs, since only 3 of 27 isolates that had thehlyA had the respective PAI. Of the virulence genes encoding adhesins (papC,papG,sfaA,sfaS, andafa),papC andpapG were the most prevalent in the strains of hemoculture. These two genes were present in 30% of our strains and were always found together in the isolates, including in commensal strains. These genes are part of the mannose-resistant P-fimbriae operon and have been associated withE. coli isolated from bacteremia [22, 23]. The PAIs IJ96, ICFT073, and IICFT073 harbor genes encoding P-fimbriae. Of the six strains of hemocultures containing the genespapC andpapG, three contained corresponding PAIs (Table 3). Meanwhile, all the commensal strains containing thepapC andpapG genes also have PAI ICFT073, showing a good correlation between them.Siderophore production is important for bacterial survival in the bloodstream. The aerobactin siderophore system is an important virulence factor that contributes to bacterial growth in host tissues and fluids where iron availability is limited [24, 25]. The aerobactin receptor (IutA) is commonly associated with extraintestinalE. coli and those isolated from bacteremia [22, 26]. In the present study,iutA was the most commonly found virulence gene, present in 65% of the isolates tested (Table 3), different from commensal which did not show this gene. PAI ICFT073 contains aerobactin genes, and 5 of the 13 strains containing theiutA gene also contained sequences similar to PAI ICFT073. Genes corresponding to other iron uptake systems were also identified:iroN, encoding the salmochelin receptor, was present in 55% of isolates; andfyuA, encoding the yersiniabactin receptor [24, 25], was present in 45% of isolates. PAI IV536 contains yersiniabactin encoding genes, and 8 of the 9 isolates exhibiting thefyuA gene contained PAI IV536 related sequences. Thus, there is a good correlation between the presence of this island and the genes encoding yersiniabactin. There was a good correlation between the commensal strains too and of the 24 isolates that contained thefyuA gene, 22 isolates had also PAI IV536.Of the genes that confer serum resistance (kpsII, K1,traT, andiss),traT, which encodes an outer membrane lipoprotein that contributes to serum resistance [27] was detected in 50% of the isolates.The invasion determinant encoded by theibe10 gene was present in 10% of the isolates, whereas theompT gene, encoding an outer membrane protease, was present in 15% of the isolates.These results demonstrate that the virulence of septicemicE. coli is not summarized by the presence of a single virulence factor, since each step in the infection process can be mediated by different virulence factors [14, 28], but the expression of the combination of virulence factors together with the imbalance between immune defenses and characteristics of the environment determines a multifactorial outcome [29].Several studies have demonstrated that isolates belonging to phylogenetic group B2 are more commonly extraintestinal pathogenic strains [3, 15, 22]. Our results demonstrated that group B2 (40%) was the most common group among theE. coli strains from hemoculture (Table 4). Strains belonging to group B2 also had the most PAI associated sequences, and 15 of the total 22 PAIs identified were present in this group. In contrast, in the commensal strain the most prevalent group was A (72.54%) and this group had a greater number of PAIs (86.95%) (Table 4). Moreover, seven of eight strains of group B2 had PAIs, against only one of six strains of group A. However, despite reports in the literature that isolates belonging to groups A and B1 are more often strictly commensal strains from the intestinal microbiota [3, 7, 15, 22], ten of the isolates sampled belonged to groups A and B1 (Table 3), demonstrating that these groups are also capable of causing systemic infection. The results of the current study indicate that isolates that are phylogenetically characterized as mainly commensal can in some cases be isolated from bloodstream infections, reinforcing the concept that virulence is associated with the presence of multiple virulence factors and is dependent on the host’s immune system [29]. The results also showed that group B2E. coli, despite being uncommon among commensal strains, can be present in intestinal flora (11.76% of our commensal strains) (Table 4), suggesting that they may act as a reservoir for bacteria that can cause extraintestinal infection [7].In previous reports, ExPEC strains from group B2 have been shown to contain more virulence factors than those from groups A and B1 [15, 22, 30]. However, our strains showed on average 4 to 5 virulence factors genes, regardless of the phylogenetic group, and despite the correlation between the presence of virulence genes and strains belonging to phylogenetic group B2, some isolates from group B2 were found to have few virulence genes, whereas some isolates from groups A and B1 had up to 8 virulence genes, while some strains of group B2 with a greater number of virulence factors had 8 virulence genes too (Table 3). Thus, although some isolates from groups A and B1 are limited in their virulence gene content and are not likely to be highly virulent, others in these groups contained multiple virulence factors genes that could contribute to extraintestinal virulence.Similarly, the virulence genes are not only associated with the PAIs, because some ExPEC also harbor virulence genes in plasmids [31]. Furthermore, PAI are located in regions of high genetic mobility, which show elements that allow recombination and, consequently, PAI rearrangement, deletion, and/or acquisition of foreign DNA [7].Given the importance of plasmids as mobile elements in the horizontal gene transfer, the plasmid profile was investigated. In this study, 14 strains showed these plasmids, which ranged from 1 to 5 plasmids per strain, and three plasmids transferred to anotherE. coli by conjugation, appearing to be conjugative plasmids. The transconjugants (sample 5, 17, and 19) received genesiutA,ompT,hlyF,iroN,traT, andiss, showing the possible presence of these virulence genes in plasmids (Table 5). Thus, the presence of transferable plasmids inE. coli isolated from hemocultures can contribute to the horizontal transfer of virulence genes to nonpathogenic isolates and interestingly the conjugative plasmids 5.2, from strain 5, hadiss,iroN,ompT, andhlyF genes, whose genes are generally present in typical APEC plasmids in a conserved virulence plasmidic (CVP) region [32]. These findings support the idea that APEC orE. coli from commercial chicken carcasses has a potential zoonotic risk, as well as serves as a reservoir for virulence genes for ExPEC strains [9, 28, 33].Table 5
Genetic characterization of ExPEC transconjugants.
Strains
iss
traT
iutA
iroN
ompT
hlyF
E. coli K12-711
−
−
−
−
−
−
E. coli 5
+
+
−
+
+
+
5.2
+
+
−
+
+
+
E. coli 17
−
+
+
−
−
−
17.1
−
+
+
−
−
−
E. coli 19
+
+
−
−
−
−
19.1
−
+
−
−
−
−
Serum resistance:iss and traT; iron uptake systems: iutA and iroN; proteases: ompT; toxin: hlyF.
Strains (donator of plasmids) = 5, 17, and 19; Tranconjugants = 5.2, 17.1, and 19.1.As ExPEC pathogenicity is due to genetic features such as virulence genes, pathogenicity islands, and plasmids associated with the virulence, the study of genetic factors is important to better understand these important pathogens. Thus, for the screening and even prevention of blood-borne diseases caused by ExPECs, further research should be conducted on the genetic features associated with the virulence of these pathogens. Although in stool samples of healthy people the presence of commensal strains is common, our results showed that the intestinal microbiota may harborE. coli of phylogenetic group B2. AlsoE. coli with PAIs and virulence genes suggest that the intestinal microbiota may act as a reservoir of ExPEC with virulence genetic factors present at theE. coli from blood stream infection.



---

*Source: 465054-2014-04-15.xml*