Extraction and partial purification of cytotoxic necrotizing factor from Escherichia coli and its effect on rabbits

Other Title(s)

استخلاص و تنقية جزئية لعامل النخر السمي لبكتريا الايشريكيا القولونية و تأثيره في الأرانب

Dissertant

al-Masudi, Haydar Fadil Jawad

Thesis advisor

al-Ubaydi, Murawij Abd al-Sattar
Hamzah, Subhi Jawad

Comitee Members

al-Jaylawi, Majid H.
Qandala, Nuha Juzif

University

University of Baghdad

Faculty

College of Science

Department

Biotechnology Department

University Country

Iraq

Degree

Master

Degree Date

2014

English Abstract

The present study concerned isolation and identification of Escherichia coli isolates associated with urinary tract infections that produce a toxin called cytotoxic necrotizing factor 1 (CNF-1).

These isolates were collected from patients attending Central Teaching Hospital of Pediatric and Al- Yarmouk Teaching Hospital.

A lot of 75 samples were collected, of which 58 (77.33%) samples show bacterial growth, 25(33.33%) of them collected from males, 17 (22.66%) with aerobic bacterial growth:12(15.99%) of them with gram negative bacteria whereas 5 (6.66%) were gram positive bacteria, while 50 (66.66 %) samples were collected from females, 41 (54.66%) with aerobic bacterial growth: 35 (46.66%) gram negative bacteria, and 6 (7.99 %) gram positive bacteria, where 35 (46.66%) isolates belonged to the family Enterobacteriaceae, E.

coli isolates form 18 (51.42%) E.

cloacae, while K.

pneumonia 6 (17.14%), 4 (11.42%) isolates respectively.

Whereas P.

mirabillis 3(8.57%), S.

marcescens 2(5.71%) and M.

morganii, C.

freundii were 1(2.85%) isolate for each one.

The sensitivity of E.

coli isolates toward study antimicrobials showed 12 (66.66%)resistant for E , CLO , CIP ,while 13 (72.22%), 11 (61.11%) , 9 ( 50%) ,8 (44.44%), and 4 (22.22%) for Amp , DA, GN, DO, TMP, AZM, AK, CTX respectively.

CNF1 has been extracted from a selected E.

coli isolates and its concentration was estimated to be 86.4μg/ml as a crude protein.

Detection of CNF1 in the crude extract was conducted in vivo by injection intradermal of mouse footpad; this showed a severe inflammation and necrosis in the footpad, and microscopically showed thickness, fibrous network and congestion of blood vessels, as well as infiltration of neutrophils in the epidermis.

The dermis layer showed mild hydropic degeneration .On the other hand ,PCR technique indicate to the foundation of chromosomal cnf1 gene in the tested isolate .The CNF1 was purified partially by using the ion exchange (DEAE) which facilitate the separation of two main peaks one of them consists of the fractions 70-98 Summary II which was farther selected.

The protein concentration was determined being 73 μg/ml using BSA stander curve.

The partially purified CNF1 revealed a single band in SDS-PAGE with molecular weight of 115kDa.

The apoptosis (in vivo) assay was determined by using flow cytometer technique.

This showed different effects; early, late and necrosis depending on the dose used (1.82, 3.65, 5.47, 7.3) μg/kg, the last two doses revealed sever effects on rabbits, while the first dose showed mild effects .The histological study of the rabbits treated with 3.65 μg/kg CNF1 revealed different changes in the liver, kidneys, and lungs ranged between severe congestion in liver and lung to paleness of kidney with change in their texture grossly.

While microscopically showed infiltration of inflammatory cells with fibrin network (pink colour), and cellular necrosis.

Main Subjects

Biology

Topics

• Iraq

No. of Pages

106

Table of Contents

• APA
• MLA
• AMA

Language

English

Data Type

Arab Theses

Record ID

BIM-601113