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

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

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

    • 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.
    • 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
    • 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
    • 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.
    • 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.
    • 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.
    • 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 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.
    • 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)*
    • 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)
    • 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)
    • 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).
    • 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.