1. PCR Detection of Virulence Factors in
Escherichia coli from
Diabetic and Non-Diabetic Patients
with Urinary Tract Infections
in Tripoli-Libya
Khalifa Sifaw Ghenghesh, Einass El-Kateb,
Nuri Berbash, Rania Abdel-Khalek,
Salwa F. Ahmed, John D. Klena
Faculty of Medicine, Tripoli University;
Faculty of Sciences; Tripoli University, Tripoli, Libya and
US Naval Medical Research Unit#3, Cairo, Egypt.

2. Introduction
• Uropathogenic Escherichia coli (UPEC) is the most
common cause of urinary tract infections (UTIs) in
humans.
• UPEC colonization of the UT is made possible by several
virulence factors (VF):
–
–
–
–
–
fimbrial adhesins >> e.g. type-1 fimbriae >> adherence
nonfimbrial adhesins >> adherence
sidrophores >> iron-acquisition systems
secreted toxins >> hemolysin
cytotoxic necrotizing factor 1 >> acts on epithelial or endothelial
cells, or monocyte-macrophages via
activation of GTP-binding proteins
– capsule-forming polysaccharides >> immune evasion

3. Introduction
• Phylogenetic groups of E. coli strains:
– Groups A and B1 >> commensal strains.
– Groups B2 and D >> mainly associated with E. coli
strains causing extraintestinal infections.
– Most VFs-containing UPECs cluster mainly either in
phylogeny group B2 or shared within groups B2 and
D
• Fluoroquinolones (e.g. ciprofloxacin):
– Drugs of choice in empirical treatment of
uncomplicated UTI.
– Studies have shown that resistance of UPEC to
ciprofloxacin and to may be associated with
decreased expression or loss of some virulence
factors

4. Introduction
• In developed countries:
– Several investigators have reported that infections
including UTIs are more frequently reported among
diabetic patients than the general population, while
others found no such difference.
– Studies have shown that hosts with predisposing
factors, such as diabetes, can acquire UTIs caused
by less virulent E. coli strains.
• In developing countries.
– Lack of information on the phylogenetic grouping
and virulence factors of UPEC from diabetic and
non-diabetic patients.

5. Objectives of the Study
• To determine the phylogenetic grouping and
the virulence factors of UPEC isolated from
diabetic and non-diabetic patients in Tripoli,
Libya using PCR techniques.

6. Methods
• Using multiplex PCR
– 48 E. coli isolates from Libyan patients with UTIs:
• 19 from diabetics and 29 from non-diabetics
• Examined for the presence of genetic determinants of
19 extraintestinal virulence factors.
• The primer pairs for the 19 virulence genes and
amplification conditions have been described
previously (Johnson & Stell 2000).
• Examined for phylogenetic grouping (A, B1, B2, or D) as
previously reported (Clermont et al., 2000).
• Reactions were performed in a GeneAmp PCR System
9700 (Applied Biosystems, Foster City, CA, USA).
• DNA sequencing was used to confirm the identity of
amplified PCR products and establish positive controls.

7. PCR detection of virulence genes in UPEC

8. Results
• Of the virulence genes examined, 3 or more
were found in 37.5% of E. coli studied.
• Only genes fimH (coding for type 1 fimbriae)
and traT (coding for serum resistance) were
both found significantly more common in E.
coli from non-diabetic than in diabetic patients
(P<0.02, OR=7.88 and P<0.00007, OR=14.38,
respectively).
• several of the VFs detected in the present study
were not reported previously in E. coli isolates
from diabetics with UTI and these include fyuA,
kpsMT II, and traT.

9. Table 1. Distribution of virulence factors (VF) among uropathogenic
Escherichia coli isolated from diabetics and non-diabetics
VF
No (%) of associated VF
Diabetics
Non-diabetics
(n=19)
(n=29)
--------------------------------------------------------------------------------------------------------------------------------------papA (P fimbriae)
0 (0.0)
0 (0.0)
papC (P fimbriae)
1 (5)
3 (10)
papEF (P fimbriae)
1 (5)
3 (10)
sfa/focD (Dr family adhesions)
2 (11)
8 (28)
focG (F1C fimbriae)
1 (5)
7 (24)
afa/draBC (Dr family adhesions)
0 (0.0)
4 (14)
bmaE (M fimbirae)
0 (0.0)
0 (0.0)
nfaE (Nonfimbrial adhesion-1)
0 (0.0)
0 (0.0)
fimH (Type 1 fimbriae)
12 (63)
27 (93)*
hylA (hemolysin)
4 (21)
7 (24)
fyuA (Yersiniabactin)
12 (63)
11 (38)
iutA (Aerobactin)
13 (68)
21 (72)
kpsMT II (Capsule synthesis group II)
7 (37)
11 (38)
kpsMT III (Capsule synthesis group III)
0 (0.0)
0 (0.0)
rfc (O4 LPS)
0 (0.0)
0 (0.0)
ibeA (Invasion)
0 (0.0)
1 (4)
cvaC (Colicin V)
0 (0.0)
1 (4)
traT (Serum resistance)
4 (21)
23 (79)*
PAI (Pathogenicity-associated island)
0 (0.0)
2 (7)
Multivirulent (>3 genes)
11 (58)
25 (86)*

10. Table 2. Distribution of virulence factors (VF) among uropathogenic Escherichia coli
isolated from diabetics and non-diabetics in relation to ciprofloxacin resistance
VF
No (%) of associated VF
Cipro-susceptible
Cipro-resistant
(n=38)
(n=10)
------------------------------------------------------------------------------------------------------------------------------------------papA (P fimbriae)
0 (0.0)
0 (0.0)
papC (P fimbriae)
4 (11)
0 (0.0)
papEF (P fimbriae)
4 (11)
0 (0.0)
sfa/focD (Dr family adhesions)
10 (26)
0 (0.0)
focG (F1C fimbriae)
8 (21)
0 (0.0)
afa/draBC (Dr family adhesions)
4 (11)
0 (0.0)
bmaE (M fimbirae)
0 (0.0)
0 (0.0)
nfaE (Nonfimbrial adhesion-1)
0 (0.0)
0 (0.0)
fimH (Type 1 fimbriae)
32 (84)*
4 (40)
hylA (hemolysin)
11 (29)*
0 (0.0)
fyuA (Yersiniabactin)
19 (50)
4 (40)
iutA (Aerobactin)
28 (74)*
4 (40)
kpsMT II (Capsule synthesis group II)
18 (47)*
0 (0.0)
kpsMT III (Capsule synthesis group III)
0 (0.0)
0 (0.0)
rfc (O4 LPS)
0 (0.0)
0 (0.0)
ibeA (Invasion)
1 (3)
0 (0.0)
cvaC (Colicin V)
1 (3)
0 (0.0)
traT (Serum resistance)
23 (61)
4 (40)
PAI (Pathogenicity-associated island)
1 (3)
1 (10)
Multivirulent (>3 genes)
34 (90)*
2 (20)

11. Phylogeny groups among UPEC isolated from diabetics and
non-diabetics and in relation to ciprofloxacin resistance
Non
Phlyogeny
Diabetics
diabetics
Ciprosusceptible
Ciproresistant
group
(n=19)
(n=29)
(n=38)
(n=10)
---------------------------------------------------------------------------------------------------------------------
A
B1
B2
D
14 (74)1
0 (0.0)
3 (16)
2 (11)
4 (14)
1 (3.4)
8 (28)
16 (55)3
10 (26)
1 (3)
10 (26)
17 (45)4
8 (80)2
0 (0.0)
1 (10)
1 (10)

12. Results
• E. coli from diabetics is significantly statistically
associated with phylogeny group A than E. coli from
non-diabetics (P<0.00003, OR=17.50).
• E. coli from non-diabetics is significantly statistically
associated with phylogeny group D than E. coli from
diabetics (P<0.002, OR=10.46).
• Ciprofloxacin-resistant E. coli is significantly
statistically associated with phylogeny group A than
ciprofloxacin-susceptible E. coli (P<0.002, OR=11.20).
• Ciprofloxacin-susceptible E. coli is significantly
statistically associated with phylogeny group D than
ciprofloxacin-resistant E. coli (P<0.05, OR=7.29).

13. Conclusion
• In developing countries, UPEC causing UTIs in
diabetics are less virulent than UPEC causing UTIs
in non-diabetics which confirms the findings of
previously published studies from developed
countries.
• Studies are needed on the prevalence of VFs and
phylogenetic groups in UPEC from diabetic patients
in developing areas.
• These studies are important in understanding the
roles of these factors in causing UTI in diabetics as
well as in non-diabetics, which in turn may lead to
development of universal vaccines to prevent such
infections.