PCR Detection of Virulence Factors in
Escherichia coli from
Diabetic and Non-Diabetic Patients
with Urinary Tract Infectio...
Introduction
• Uropathogenic Escherichia coli (UPEC) is the most
common cause of urinary tract infections (UTIs) in
humans...
Introduction
• Phylogenetic groups of E. coli strains:
– Groups A and B1 >> commensal strains.
– Groups B2 and D >> mainly...
Introduction
• In developed countries:
– Several investigators have reported that infections
including UTIs are more frequ...
Objectives of the Study
• To determine the phylogenetic grouping and
the virulence factors of UPEC isolated from
diabetic ...
Methods
• Using multiplex PCR
– 48 E. coli isolates from Libyan patients with UTIs:
• 19 from diabetics and 29 from non-di...
PCR detection of virulence genes in UPEC
Results
• Of the virulence genes examined, 3 or more
were found in 37.5% of E. coli studied.
• Only genes fimH (coding for...
Table 1. Distribution of virulence factors (VF) among uropathogenic
Escherichia coli isolated from diabetics and non-diabe...
Table 2. Distribution of virulence factors (VF) among uropathogenic Escherichia coli
isolated from diabetics and non-diabe...
Phylogeny groups among UPEC isolated from diabetics and
non-diabetics and in relation to ciprofloxacin resistance
Non
Phly...
Results
• E. coli from diabetics is significantly statistically
associated with phylogeny group A than E. coli from
non-di...
Conclusion
• In developing countries, UPEC causing UTIs in
diabetics are less virulent than UPEC causing UTIs
in non-diabe...
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PCR detection of virulence factors in Escherichia coli-Tripoli-Libya

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PCR detection of virulence factors in Escherichia coli-Tripoli-Libya

  1. 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. 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. 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. 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. 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. 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. 7. PCR detection of virulence genes in UPEC
  8. 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. 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. 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. 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. 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. 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.
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