Virulence of uropathogenic Escherichia coli isolates and their potential to be diarrheagenic Marium Khaleque1, Selina Akter2, Humaira Akhter1, Sirajul Islam Khan1, Anowara Begum1 Affiliation : 1 Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh. 2 Department of Microbiology, Jessore University of Science and Technology, Jessore- 7408, Bangladesh. Presented By: Department of Microbiology Marium Khaleque University of Dhaka

Introduction

Escherichia coli UTI UPEC Virulence Factors of DEC Family: Enterobacteriaceae common inhabitant of the gastrointestinal tract of humans and animals. Some strains of E. coli can diverge from their commensal cohorts, taking on a more pathogenic nature. These strains acquire specific virulence factors which confer an increased ability to adapt to new niches and allow them to cause a broad spectrum of disease The sub-set of genes involved in the subversion of host responses and attacking of host cell machinery makes each pathovar distinct

Virulence Factors of DEC UTI UPEC Virulence Factors of DEC Escherichia coli E. coli can be classified into following groups: UPEC (Uropathogenic E. coli) collected from UTI patient Commensal Pathogenic The commensal E. coli is the nonpathogenic and often considered to be the normal inhabitant of the gastrointestinal tract of warm-blooded animals Diarrheagenic Extraintestinal UPEC NMEC can cause infection in the gastrointestinal tract EPEC EHEC ETEC EIEC EAEC DAEC is the group capable of infecting sites other than intestine

Virulence Factors of DEC UTI UPEC Virulence Factors of DEC Escherichia coli A urinary tract infection (UTI) is an infection in any part of the urinary system including kidneys, ureters, bladder and urethra. E. coli is responsible for more than 80% of the UTI cases Causative agents E. coli Klebsiella spp Enterobacter spp Proteus Pseudomonas Staphylococcus saprophyticus

Aims and Objectives Assessment of virulence properties of uropathogenic E. coli (UPEC), isolated from urine samples of UTI patients. Whether these strains share the virulence properties with diarrheagenic E. coli by monitoring the presence of five different diarrheagenic E. coli virulence traits in these UPEC strains.

Virulence Factors of DEC autotransporter toxin Escherichia coli UTI UPEC Virulence Factors of DEC E. coli that can enter, colonize urinary tract and cause infection are known as uropathogenic E. coli Virulence factors of UPEC Type 1 fimbriae virulence factors associated with bacterial cell surface α-haemolysin secreted and exported to the site of action P fimbriae CNF1 S fimbriae CDT afimbrial adhesins autotransporter toxin

Virulence Factors of DEC Escherichia coli UTI Virulence Factors of DEC UPEC vt2 pCVD eltB vt1 estA EHEC EPEC ETEC EIEC EAEC eaeA bfpA ial

Identification of isolates Work plan Microscopy Identification of isolates Use of selective and differential media Biochemical profiling Preliminary screening Biochemical screening Identification using the fluorogen MUG Biochemical Identification Observation of UTI - associated virulence properties Observation of genes associated with diarrheagenic E. coli virotypes by multiplex PCR assay hemolysis activity Biofilm formation UTI associated adhesin genes by multiplex PCR assay

Result

Identification of isolates Among 71 isolates 56 (78.8%) isolates were identified as E. coli Biochemical Screening Microscopy selective and differential media MUG test

Observation of UTI - associated virulence properties Hemolysis activity Among 56 isolates 5 isolates gave clear zone of β-hemolysis around the colony. No α-haemolysis was found among the E. coli isolates. β-hemolysis γ- hemolysis

Observation of UTI - associated virulence properties Biofilm formation assay Among 56 isolates 45% isolate showed the capability of biofilm formation 16 gave significant biofilm formation ability and 9 gave moderate level of biofilm formation ability in-vitro. Naves et al., 2008

Observation of UTI - associated virulence properties Detection of UTI associated adhesin genes by multiplex PCR assay Type I fimbriae Among 56 E. coli isolates 27% was fim1 (+)ve P fimbriae 43% was papC (+)ve 11% was afa (+)ve No isoletes found (+)ve for sfa adhesin genes Fig: Agarose gel elctrophoresis showing PCR amplification product of papC (328 bp) gene and afa (750 bp), fim1 (700 bp) gene and No band appeared for sfa adhesin genes

Potentiality of UPEC isolates to be diarrheagenic Detection of diarrheagenic virulence genes by multiplex PCR assay All the 56 UPEC strains were subjected to multiplex PCR for the detection of heat stable toxin (estA) Only 7 isolates was carrying DEC virulence gene heat labile toxin (eltB) bundle forming pilli (bfpA), EA (pCVD) gene Verotoxin 2 (vt2) Verotoxin 1 (vt1), Eae (eaeA) Invasive associated locus (ial)

Detection of diarrheagenic virulence genes by multiplex PCR assay Potentiality of UPEC isolates to be diarrheagenic Detection of diarrheagenic virulence genes by multiplex PCR assay All the 56 UPEC strains were subjected to multiplex PCR for the detection of 2 ETEC heat stable toxin (estA) Only 7 isolates was carrying DEC virulence gene heat labile toxin (eltB) 1 ETEC EPEC bundle forming pilli (bfpA), 1 EA (pCVD) gene EAEC 1 Verotoxin 2 (vt2) EHEC 1 Verotoxin 1 (vt1), Eae (eaeA) EIEC Invasive associated locus (ial) 1

Comparison with other study Similar study in Brazil, Iran, Denmark, Germany 16 out of 225 UPEC strain Conclusion In Bangladesh, isolates of UPEC have been found sharing virulence property with Diarrheagebic E. coli But these UPEC strains were found to share with different DEC virulence properties ….NOT limited to EAEC In our study, we have found almost equal no. of UPEC strains carrying virulence gene of ETEC, EHEC, EPEC, EIEC or EAEC. Among these ETEC property was found in higher no. of UPEC strains. Brazil aat seq of EAEC 2007 13 out of 138 UPEC strain Iran EAEC property 2014 increased adherence to human bladder epithelial cell EAEC virulence gene in UPEC In the presence of EAEC gene OUTBREAK Copehegan, Denmark in 1991 10.8% of 265 UPEC strains Germany EAEC property 2005-2006

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Patient information Cultures were collected from 71 patient . Among the patients 47 (66.2%) were female and the rest 24 (33.8%) were male, indicating higher occurrence of UTI in females in a random sample Fig. Age and sex distribution pattern of 71 UTI patients of the study.