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Activity of Pimpinella anisum and Hydnora abissynica Against Associated with Child Diarrhoea

By Abd Elfadil Ibraheem Ali Mohammed B.Sc. Omdurman Islamic University (٢٠٠٢)

Supervisor

Dr. Suliman Mohammed El Hassan

A thesis submitted to University of Khartoum in partial fulfillment of requirement for Master degree of Science in Microbiology

Department of Microbiology Faculty of Veterinary Medicine University of Khrtoum (٢٠٠٦) April ﻗﺎل ﺘﻌﺎﻟﻲ:

ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﺣﻤﻦ ﺍﻟﺮﺣﻴﻢ

( وﻋﻠﻢ ﺁدم اﻷﺳﻤﺎء آﻠﻬﺎ ﺛﻢ ﻋﺮﺿﻬﻢ ﻋﻠﻰ اﻟﻤﻼﺋﻜﺔ ﻓﻘﺎل أﻧﺒﺆﻧﻲ ﺑﺄﺳﻤﺎء هﺆﻻء إن آﻨﺘﻢ ﺻﺎدﻗﻴﻦ* ﻗﺎﻟﻮا ﺳﺒﺤﺎﻧﻚ ﻻ ﻋﻠﻢ ﻟﻨﺎ إﻻ ﻣﺎ ﻋﻠﻤﺘﻨﺎ

إﻧﻚ أﻧﺖ اﻟﻌﻠﻴﻢ اﻟﺤﻠﻴﻢ * ).

ﺼﺩﻕ ﺍﷲ ﺍﻟﻌﻅﻴﻡ

ﺴﻭﺭﺓ ﺍﻟﺒﻘﺭﺓ (٣١- ٣٢)

PREFACE

This work was carried out at The Departmet of Microbiology,Fuculty of Veterinary Medicine,Univercity of Khartoum, Sudan under supervissor of Dr. Suliman Mohammed El Hassan.

DEDICATION

To soul of my father, To my mother, brothers, and sisters…

ACKNOWLEDGMENTS

I would like to express my sincere gratitude and indebtedness to my supervisor Dr. Suliman Mohammed El Hassan the head of Microbiology Department, Faculty of Veterinary Medicine, University of Khartoum, without him this piece of work have never seen light. Special gratitude goes to staff of Faculty of Veterinary Medicine for their kind assistance throughout the study. My special acknowledgment extended to all technicians, technical assistance and working staff in the Microbiology Department, Faculty of Veterinary Medicine, University of Khartoum, Abd Elazeez, Fawzia, Mona, Hashim, Elyas, Abdalla, Abd Elazeem and Sorria, for their great help. Also I would like to thank the directors and staff of Omdurman Pediatric Teaching Hospital for their great assistance in collection of samples. My thanks are due to the staff of the National Research Center, Dr. Hassan Elsubky, Mudasir and Hosham for helping me to prepare the medicinal plants exrracts. Finally, I would like to thank Yousif Mostafa Elibead , folkloric medicine expert for his continuous help as he gave me information about medicinal plant that related to my study.

TABLE OF COTENS PREFACE…………………………………………………………… III DEDICATION………………………………………………………… IV ACKNOWLEDGMENTS…………………………………………… V TAPLE OF CONTENT………………………………………………. VI LIST OF TABLES…………………………………………………… XIV LIST OF FIGUERS………………………………………………… XVI ABSTRACT…………………………………………………………… XVII ARABIC ABSTRACT……………………………………………… XIX INTRODUCTION…………..………………………………………… XXI ١ …………………………CHAPER ONE: LETRETUER REVIEW ١ …………………………………………………………Diarrhoea ١٫١ ١٫١٫٢ ١ Definition…………………………………………………………

١,١,٣ ١ Classification……………………………………………………... Acute ١٫١٫٣٫١ ١ diarrhoea...... Chronic ١٫١٫٣٫٢ ٢ diarrhoea...... ٢ ...... Causative agent of diarrhoea ١٫١٫٤ ٣ ...... Mechanisms ١٫١٫٥ Mucosal ١٫١٫٥٫١ ٣ adherence...... ٣ ...... Mucosal invasion .١٫١٫٥٫٢ ٤ ...... Toxin production .١٫١٫٥٫٣ ٤ ...... Magnitude of the problem ١٫١٫٦ Transmission and spread of ١٫١٫٧ ٥ infection...... ٦ ...... Diarrhoea in Sudan ١٫١٫٨ ٧ ...... ١٫٢ ٧ ...... and classification of Enterobacteriaceae ١٫٢٫١ ٨ ...... Esherichia ١٫٢٫٣ ٨ ...... Pathogenisis of E. coli ١ ١٫٢٫٣ Enterotoxogenic E. coli ١٫٢٫٣٫١٫١ ٨ (ETEC)...... Enteroinvasive E. coli ١٫٢٫٣٫١٫٢ ١٠ (EIEC)...... Enteropathogenic E. coli ١٫٢٫٣٫١٫٣ ١١ (EPEC)...... Verocytotoxic E. coli ١٫٢٫٣٫١٫٤ ١٢ (VTE)...... Entero – aggregative E. coli ١٫٢٫٣٫١٫٥ ١٣ (EAggEC)...... Diffuse adherent E. coli ١٫٢٫٣٫١٫٦ ١٤ (DAEC)...... Viability of E ١٫٢٫٣٫٢ ١٤ coli...... ١٤ ...... Shigella specsies ١٫٢٫٤ ١٫٢٫٤٫١ ١٤ Description...... ١٫٢٫٤٫٢ ١٤ Classification...... ١٥ ...... Incidence and source of shigella infection ١٫٢٫٤٫٣ ١٫٢٫٤٫٤ ١٥ Pathogenesis...... Proteus species ١٫٢٫٥ ١٦ ... Normal ١٫٢٫٥٫١ ١٦ habitat...... General ١٫٢٫٥٫٢ ١٦ characteristic...... Antibiotic ١٫٢٫٥٫٣ ١٧ sensitivity...... Providencia ١٫٢٫٦ ١٧ species...... Klebsiella ١٫٢٫٧ ١٧ species...... Kluvera ١٫٢٫٨ ١٨ species...... Cedecea ١٫٢٫٩ ١٨ species...... Tatumela ١٫٢٫١٠ ١٨ species...... Enterobacter ١٫٢٫١١ ١٨ species...... Morganella ١٫٢٫١٢ ١٩ species...... Edwardsiella ١٫٢٫١٣ ١٩ species...... Citrobacter ١٫٢٫١٤ ٢٠ species...... Hafnia ١٫٢٫١٫١٥ ٢٠ species...... Serratia ١٫٢٫١٦ ٢١ species...... ٢٢ ...... Salmonella ١٫٢٫١٧ ١٫٢٫١٧٫١ ٢٢ Description...... Classification and ١٫٢٫١٧٫٢ ٢٢ nomenclature...... ١٫٢٫١٧٫٣ ٢٣ Pathogenicity...... Medicinal ١٫٣ ٢٤ plants...... ٢٤ ...... (Anise (Pimpinella anisum ١ ١٫٣ ٢٧ ...... Hydnora abissynica ١٫٣٫٢ ٣١ ...... CHAPTER TWO: MATERIALS AND METHODS ٣١ ...... Asepsis and sterilization ٢٫١ ٣١ ٢٫١٫١ Flaming...... ٣١ ...... Asepsis and sterilization ٢٫١ ٣١ ٢٫١٫١ Flaming...... ٣١ ...... Red heat ٢٫١٫٢ ٣١ Hotair ٢٫١٫٣ ...... oven٢ ٣١ Steaming at ٢٫١٫٤ ...... C°١٠٠ ٣١ Moist heat ٢٫١٫٥ (autoclave)...... ٣١ ٢٫١٫٦ Irradiation...... ٣١ ...... Disinfection ٢٫١٫٧ ٣١ ...... Reagents and indicators ٢٫٢ ٣١ ٢٫٢٫١ Reagents...... ٣٢ Sodium chloride /normal ٢٫٢٫١٫١ saline...... ٣٢ Hydrogen peroxide ٢٫٢٫١٫٢

...... (H٢O٢) ٣٢ Potassium ٢٫٢٫١٫٣ hydroxide...... ٣٢ Kovac’s ٢٫٢٫١٫٤ reagent...... ٣٢ Lead ٢٫٢٫١٫٥ acetate...... ٣٢ Potassium hydroxide and hydrochloric ٢٫٢٫١٫٦ acid...... ٣٢ Methyl red ٢٫٢٫١٫٧ solution...... ٣٢ Alpha – naphthol ٢٫٢٫١٫٨ solution...... ٣٢ Tetramethyl – p – phenyline diamine ٢٫٢٫١٫٩ diahyrochloride...... ٣٣ ٢٫٢٫٢ Indicators...... ٣٣ Bromothymol ٢٫٢٫٢٫١ blue...... ٣٣ Andrade's ٢٫٢٫٢٫٢ indicator...... ٣٣ Bromocrysol ٢٫٢٫٢٫٣ purple...... ٣٣ Gram stains ٢٫٢٫٢٫٤ reagents...... ٣٣ Lugol’s ٢٫٢٫٢٫٤٫١ iodine...... Crystal ٢٫٢٫٢٫٤٫٢ ٣٤ violet...... Acetone – alcohol ٢٫٢٫٢٫٤٫٣ ٣٤ decolorizer...... Diluted carbol ٢٫٢٫٢٫٤٫٤ ٣٤ fuchsin...... Equipment ٢٫٣ ٣٥ used...... ٣٥ ...... Antibiotic used ٢٫٤ Culture ٢٫٥ ٣٥ media...... ٣٥ ...... Liquid media ٢٫٥٫١ MR test medium (Glucose – phosphate ٢٫٤٫١٫١ ٣٥ medium)...... V P test medium (glucose – phosphate ٢٫٥٫١٫٢ ٣٦ medium)...... Peptone ٢٫٥٫١٫٣ ٣٦ water...... ٣٦ Nitrate ٢٫٥٫١٫٤ medium...... ٣٦ ...... Nutrient broth ٢٫٥٫١٫٥ ٣٦ ...... Peptone water sugar ٢٫٥٫١٫٦ -Selenite- F ٢٫٥٫١٧ ٣٧ broth...... ٣٧ ...... Semi solid media ٢٫٥٫٢ ٣٧ ...... Oxidation – Fermentation (O/ F) medium ٢٫٥٫٢٫١ ٣٧ ...... Carry – Blair transport medium ٢٫٥٫٢٫٢ Motility medium – Gragie tube ٢٫٥٫٢٫٣ ٣٨ medium...... ٣٨ ...... Solid media ٢٫٥٫٣ MacConkey's ٢٫٥٫٣٫١ ٣٨ agar...... ٣٨ ...... Diagnostic sensitivity test (DST) agar ٢٫٥٫٣٫٢ Kligller's iron agar ٢٫٥٫٣٫٣ ٣٩ (KIA)...... Nutrient ٢٫٥٫٣٫٤ ٣٩ agar...... ٤٠ ...... Nutrient gelatin ٢٫٥٫٣٫٥ SS (Salmonella – Shigella) agar (M ٢٫٥٫٣٫٦ ٤٠ ...... (١٠٨ Simmon’s citrate ٢٫٥٫٣٫٧ ٤٠ agar...... Urea agar ٢٫٥٫٣٫٨ ٤١ base...... Ammnonium salt sugars ٢٫٥٫٣٫٩ ٤١ (ASS)...... ٤١ ...... Collection and transport of sample ٢٫٦ Culture of ٢٫٧ ٤٢ specimens...... Microscopic ٢٫٨ ٤٢ examination...... ٤٢ ...... Identification of bacteria ٢٫٩ ٤٣ ...... Biochemical method ٢٫١٠ Catalase ٢٫١٠٫١ ٤٣ test...... Citrate ٢٫١٠٫٢ ٤٣ test...... Urease ٢٫١٠٫٣ ٤٣ test...... Oxidase ٢٫١٠٫٤ ٤٣ test...... Sugar fermentation ٢٫١٠٫٥ ٤٤ test...... Indole ٢١٠٫٦ ٤٤ test...... (Oxidation – fermentation (O/F ٢٫١٠٫٧ ٤٤ test...... (Methyl red (MR ٢٫١٠٫٨ ٤٤ test...... Gelatin hydrolysis ٢٫١٠٫٩ ٤٥ test......

Hydrogen sulphide (H٢S) production ٢٫١٠٫١٠ ٤٥ test...... Nitrate reduction ٢٫١٠٫١١ ٤٥ test...... (Voges – Proskaur's(VP ٢٫١٠٫١٢ ٤٦ test...... Motility ٢٫١١ ٤٦ test...... ٤٦ ...... Staining method ٢٫١٢ ٤٧ ...... Plants extraction methods ٢٫١٣ Extraction of Hydnora ٢٫١٣٫١ ٤٧ abissynica...... Water ٢٫١٣٫١٫١ ٤٧ extraction...... Petroleum ether ٢٫١٣٫١٫٢ ٤٧ extraction...... ٤٨ ...... Anise (Pimpinella anisum) volatile oil extrataction .٢٫١٣٫٢ ٤٨ ...... Cup plate method .١٫١٤ ٥٠ ...... CHAPTER THREE: RESLUT Isolation and ٣٫١ ٥٠ identification...... ٥٠ ...... Main findings ٣٫٢ ٣٫٢٫١ ٥٠ isolates...... Cultural ٣٫٢٫١٫١ ٥٠ characteristics...... Microscopic ٣٫٢٫١٫٢ ٥١ Examination...... Biochemical ٣٫٢٫١٫٣ ٥١ reaction...... Shigella species ٣٫٢٫٢ ٥١ isolates...... ٣٫٢٫٢٫١Cutural ٥١ characteristics...... Microscopic ٣٫٢٫٢٫٢ ٥١ Examination...... ٣٫٢٫٢٫٣Biochemical ٥١ reaction...... Citrobactor freundii ٣٫٢٫٣ ٥١ isolates...... Cultural ٣٫٢٫٣٫١ ٥٢ characteristics...... Microscopic ٣٫٢٫٣٫٢ ٥٢ examination...... Biochemical ٣٫٢٫٣٫٣ ٥٢ reaction...... Enterobacter species ٣٫٢٫٤ ٥٢ isolates...... Cultural ٣٫٢٫٤٫١ ٥٢ Characteristics...... Microscopic ٣٫٢٫٤٫٢ ٥٢ examination...... ٣٫٢٫٤٫٣Biochemical ٥٢ reactions...... ٥٢ ...... Pseudomonas species isolate ٣٫٢٫٥ ٥٢ ...... The cultural Characteristics ٣٫٢٫٥٫١ Microscopic ٣٫٢٫٥٫٢ ٥٣ examination...... Biochemical ٣٫٢٫٥٫٣ ٥٣ reaction...... Proteus species ٣٫٢٫٦ ٥٣ isolates...... Cultural ٣٫٢٫٦٫١ ٥٣ characteristics...... Microscopic ٣٫٢٫٦٫٢ ٥٣ examination...... Biochemical ٣٫٢٫٦٫٣ ٥٣ reactions...... Klebsiella species ٣٫٢٫٧ ٥٤ isolates...... Cultural ٣٫٢٫٧٫١ ٥٤ characteristics...... Microscopic ٣٫٢٫٧٫٢ ٥٤ examination...... Biochemical ٣٫٢٫٧٫٣ ٥٤ reaction...... Salmonella species ٣٫٢٫٨ ٥٤ isolates...... Cultural ٣٫٢٫٨٫١ ٥٤ characteristics...... Microscopic ٣٫٢٫٨٫٢ ٥٥ examination...... Biochemical ٣٫٢٫٨٫٣ ٥٥ reaction...... Providencia alcalifacient ٣٫٢٫٩ ٥٥ isolates...... Cultural ٣٫٢٫٩٫١ ٥٥ characteristics...... Microscopic ٣٫٢٫٩٫٢ ٥٥ examination...... Biochemical .٣٫٢٫٩٫٣ ٥٥ reactions...... Serratia species ٣٫٢٫١٠ ٥٦ isolates...... Cultural ٣٫٢٫١٠٫١ ٥٦ characteristics...... Microscopic ٣٫٢٫١٠٫٢ ٥٦ examination...... Biochemical ٣٫٢٫١٠٫٣ ٥٦ reactions...... In vitro antibacterial activity of some ٣٫٣ ٥٦ antibiotics…………………... In vitro antibacterial activity of plant ٣٫٤ ٥٧ .………………………extract

In vitro antibacterial activity of volatile extract oil of ٣٫٤٫١ ٥٧ anise……...

In vitro antibacterial activity of water extracts of Hydnora ٣٫٤٫٢ ٥٨ ………………………………………………………………………abissynica

٧٧ ...... CHAPTER FOUR: DISCSSION ٨٣ ...... …………………………………………… CONCLUSIONS ٨٤ ...... RECOMMENDATIONS ٨٥ ...... REFRENCES

LIST OF TABLES

Bacterial species isolated from children diarrhoeal samples :١ Table ٥٩ .………collected from patients in Omdurman Pediatric Teaching Hospital a: Characters and biochemical reactions of bacteria isolated from ٢ Table child diarrheal samples collected from patients in Omdurman Pediatric ٦٠ ...... ……………………………………………Teaching Hospital b: Characters and biochemical reactions of bacteria isolated from ٢ Table child diarrheal samples collected from patients in Omdurman Pediatric ٦١ …………………………………………………………Teaching Hospital Antimicrobial activity of different antibiotics against bacteria :Table٣ isolated from child diarrhoeal samples collected from patients in ٦٢ ...… ...……………………………Omdurman Pediatric Teaching Hospital Antimicrobial activity of three different concentrations of :٤ Table volatile oil extracts of Pimpinella anisum against bacteria isolated from child diarrhoeal samples collected from patients in Omdurman Pediatric ٦٤ .……………………………………………………………………Hospital Antimicrobial activity of water extract without heating of :٥ Table Hydnora abissynica against bacteria isolated from child diarrhoeal samples collected from patients in Omdurman Pediatric Teaching ٦٥ .……………………………………………………………………Hospital (٨٠oC-٧٠) Antimicrobial activity of water extract with heating at :٦ Table ٤h of Hydnora abissynica against bacteria isolated from child for diarrhoeal samples collected from patients in Omdurman Pediatric ٦٦ ..…………………………………………………………Teaching Hospital oC) and dried water ٨٠-٧٠) Antimicrobial activity of heated :٧ Table concentrations against ٪١٠ and ٪٢٠ extracts of Hydnora abissynica at bacteria isolated from child diarrhoeal samples collected from patients in ٦٧ ...…………………………………Omdurman Pediatric Teaching Hospital volatile oil of anise ٪٠,١ and ٪١,٠ ,٪١٠ Antimicrobial activity of :٨ Table (P.anisum) against bacteria isolated from child diarrhoeal sample ٦٨ .………collected from patients in Omdurman Pediatric Teaching Hospital

Antimicrobial activity of H. abissynica water extracts against :٩ Table bacteria isolated from child diarrhoeal samples collected from patients in

٦٩ ...... Omdurman Pediatric Teaching Hospital concentration of ٪١٠,Antibacterial activity of six antibiotics :Table١٠ dried water extractact of H.abissynica ٪٢٠ volatile oil of P. anisum and against bacteria isolated from child diarrhoeal samples collected from ٧٠ ...... … patients in Omdurman Pediatric Teaching Hospital

LIST OF FIGURES

٢٨ …………………………………………Anise whole plant :١٫١ Figure ٢٩ ...……………………(Anise (Pimpinella anisum)fruit(seeds :١٫٢ Figure ٣٠ …..……………………………………………Hydnora abissynica :٢ Figure Bacterial species isolated from child diarrhoeal samles :٣ Figure ٧١ .……collected from patients in Omdurman Pediatric Teaching Hospital .Antimicrobial activity of six antibiotics, P.anisum and H :٤ Figure abissynica to bacteria isolated from child diarrhoeal samples collected from patients in Ondurman Pediatric Teaching ٧٢ .…………………………………………………………………Hospital Salmonella subgenus IV on MacConkey's :٥ Figure ٧٣ agar………………. ٧٣ ……………… on MacConkey's agar :٦ Figure Escherichia coli on MacConkey's :٧ Figure ٧٤ agar……………………….. ٧٤ ...…………… on nutreint agar :٨ Figure Sensitivity of Escherichia coli to six antibiotics(Nalidixtc : ٩ Figure acid, Gentamycin, Streptomysin, Ampicillin, Tetracycline and ٧٨ ..………………………………………………………………(Penicillin Sensitivity of Escherichia coli to three concentrations of : ١٠ Figure dried water extract of ٪٢٠ volatile oil of anise (Pimpinella anisum) and ٧٩ ...... ………………………………………………Hydnora abissynica

ABSTRACT

The main objective of this study was to determine bacteria associated with child diarrhoea, and to testing the isolates sensitivity to some antibiotics that commonly used for treatment of child diarrhoea and two medicinal plant used in folkloric medicine for treatment of child diarrhoea. A total of fifty-one faecal samples were collected from children suffering from acute watery and bloody diarrhoea at Omdurman Pediatric Teaching Hospital. The collected faecal samples were cultured onto and into deferential and selective media (MacConkey's agar and Selenite-F-broth) using streaking method for solid medium and inoculation for broth medium. ٣٧oC overnight. Then The streaked and inoculated media were incubated at the isolates were subcultured on Salmonella- Shigella(S.S) agar from the Selenite-F-broth. The primary cultures were purified and then identified. Sensitivity of the isolated bacteria to six antibiotics and two medicinal plant were examined. Eighty- nine bacteria strains were isolated from the faecal samples examined. All isolates had the same Gram's reaction and all of them were Gram-negative and belonged to Enterobacteriaceae family except one isolate. of the isolates were Escherichia coli ٣٦ Biochemical tests revealed associated with child (٪٧٠٫٥) indicating E.coli was the predominant isolate Enterobacter (٪١٧٫٧) ٩,Serratia polymuthica (٪١٧٫٧) diarrhoea. Nine

٥ ,Serratia marcescence (٪٩٫٨)٥ ,Klebsiella pneumoniae (٪١٣,٧) ٧ ,cloaceae

Proteus (٪٥٫٩) ٣ , (٪٧٫٨) ٤ , (٪٩٫٨)

Providencia (٪٥٫٩) ٣ , (٪٥٫٩) ٣ ,٣ vulgaris biogroup ,Salmonella subgenus III (٪٢) ١ ,Shigella dysentriae (٪٢) ١ ,aclalifacient intermedia were isoated. In (٪٢)١ Salmonella subgenus IV and (٪٢)١ .Pseudomonas aeruginosa was isolated (٪٢) ١ addition The antibacterial activity of six antibiotics and two medicinal plant (Pimpinella anisum and Hydnora abissynica) extracts were examined against sixty- seven different isolates by cup- plate method. concentration of volatile oil extractions of ٪١٠ ,The results revealed .of the isolates (٪٩٥٫٥) Pimpinella anisum inhibited the growth of sixty- four ٪٠٫١ and ٪١ of isolates were inhibited at (٪٨٥٫١)٥٧ and(٪٩٤) Sixty-three concentration of volatile oil of anise respectivelly. All water extracts of Hydnora abissynica did not inhibit the growth of any isolates examined. The antibiotics examined in this study varied in their ability to inhibit the growth of the isolated bacteria. Nalidixic acid was the most

isolates tested, while Gentamycin ٪٩٤ effective one, it inhibited the growth of

of isolate tested and they ٪٩٢,٥ was the second and inhibited the growth of

of the tested ٪٩١,٠ followed by streptomycin which ihibited the growth of isolates , but Penicillin was the least effective and inhibited the growth of only

.of isolated bacteria tested (٪١٣,٤) ٩

ﻤﻠﺨﺹ ﺍﻷﻁﺭﻭﺤﺔ

ﻜﺎﻥ ﺍﻟﻐﺭﺽ ﺍﻻﺴﺎﺴﻲ ﻤﻥ ﺍﻟﻘﻴﺎﻡ ﺒﻬﺫﺓ ﺍﻟﺩﺭﺍﺴﺔ ﺘﺤﺩﻴﺩ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻤﺼﺎﺤﺒﺔ ﻹﺴﻬﺎﻻﺕ ﺍﻷﻁﻔﺎل

ﻭﺇﺨﺘﺒﺎﺭ ﺤﺴﺎﺴﻴﺔ ﻫﺫﺓ ﺍﻟﺠﺭﺍﺜﻴﻡ ﻟﺒﻌﺽ ﺍﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﺤﻴﻭﻴﺔ ﺍﻟﺘﻲ ﺘﺴﺘﺨﺩﻡ ﻓﻲ ﻋﻼﺝ ﺇﺴﻬﺎﻻﺕ ﺍﻷﻁﻔﺎل

ﻭﺒﻌﺽ ﺍﻟﻨﺒﺎﺘﺎﺕ ﺍﻟﻁﺒﻴﺔ ﺍﻟﺘﻲ ﺘﺴﺘﺨﺩﻡ ﻓﻲ ﺍﻟﻁﺏ ﺍﻟﺸﻌﺒﻲ ﻜﻌﻼﺝ ﻹﺴﻬﺎﻻﺕ ﺍﻷﻁﻔﺎل.

ﺠﻤﻠﺔ ﻭﺍﺤﺩ ﻭﺨﻤﺴﻭﻥ ﻋﻴﻨﺔ ﻓﺴﺤﺔ ﺘﻡ ﺍﻟﺤﺼﻭل ﻋﻠﻴﻬﺎ ﻤﻥ ﺃﻁﻔﺎل ﻴﻌﺎﻨﻭﻥ ﻤﻥ ﺇﺴﻬﺎﻻﺕ ﻤﺎﺌﻴﺔ

ﻭﺩﻤﻭﻴﺔ ﺤﺎﺩﺓ ﻤﻥ ﻤﺴﺘﺸﻔﻲ ﺃﻡ ﺩﺭﻤﺎﻥ ﺍﻟﺘﻌﻠﻴﻤﻲ ﻟﻸﻁﻔﺎل. ﺍﻟﻁﺭﻴﻘﺔ ﺍﻟﺘﻲ ﺍﺴﺘﺨﺩﻤﺕ ﻟﻌﺯل ﻭﻤﻌﺭﻓﺔ ﻫﻭﻴﺔ

ﺍﻟﺠﺭﺍﺜﻴﻡ ﻜﺎﻨﺕ ﻋﻥ ﻁﺭﻴﻕ ﺯﺭﺍﻋﺔ ﺍﻟﻔﺴﺤﺔ ﻓﻲ ﺒﻴﺌﺎﺕ ﺘﻔﺭﻴﻘﻴﺔ ﻭﺇﻨﺘﻘﺎﺌﻴﺔ ﻤﺜل ﺃﺠﺎﺭ ﺍﻟﻤﺎﻜﻭﻨﻜﻲ ﻭﺒﻴﺌﺔ

ﺍﻟﺴﻼﻨﺎﻴﺕ ﻤﺴﺘﺨﺩﻤﻴﻥ ﻁﺭﻴﻘﺔ ﺍﻟﺘﺨﻁﻴﻁ ﻓﻲ ﺍﻟﺒﻴﺌﺎﺕ ﺍﻟﺼﻠﺒﺔ ﻭﻁﺭﻴﻘﺔ ﺍﻟﺘﻠﻘﻴﺢ ﻓﻲ ﺍﻟﺒﻴﺌﺎﺕ ﺍﻟﺴﺎﺌﻠﺔ، ﻭﻤﻥ ﺜﻡ

ﺘﻡ ﺘﺤﻀﻴﻥ ﻫﺫﻩ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻤﺯﺭﻋﺔ ﻓﻲ ﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ 37ْ ﻡ ﻟﻤﺩﺓ ٢٤ ﺴﺎﻋﺔ. ﻤﻥ ﺒﻴﺌﺔ ﺍﻟﺴﻼﻨﻴﺕ ﻴﺘﻡ

ﺍﻋﺎﺩﺓ ﺍﻟﺘﺯﺭﻴﻊ ﻋﻠﻲ ﺃﺠﺎﺭ ﺍﻟﺴﺎﻟﻤﻭﻨﻴﻼ ﻭﺍﻟﺸﻴﻘﻼ. ﺍﻟﻌﺯﻻﺕ ﺍﻻﻭﻟﻴﺔ ﺘﻤﺕ ﺘﻨﻘﻴﺘﻬﺎ ﻭ ﺘﻌﺭﻴﻔﻬﺎ ﻭ ﺍﺠﺭﻱ

ﺍﺨﺘﺒﺎﺭﺤﺴﺎﺴﻴﺔ ﻫﺫﺓ ﺍﻟﻌﺯﻻﺕ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ ﻟﺴﺘﺔ ﻤﻥ ﺍﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﺤﻴﻭﻴﺔ ﻭﻨﺒﺎﺘﻴﻥ ﻁﺒﻴﻴﻥ ﻭ ﻫﻤﺎ ﺍﻟﻴﻨﺴﻭﻥ ﻭ

ﺍﻟﺘﺭﺘﻭﺱ.

ﺘﻡ ﺍﻟﺤﺼﻭل ﻋﻠﻲ ﺘﺴﻊ ﻭﺜﻤﺎﻨﻭﻥ ﻋﺯﻟﺔ ﻤﻥ ﺠﻤﻠﺔ ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﺘﻲ ﺘﻡ ﺯﺭﻋﻬﺎ.ﺍﻟﻨﺘﺎﺌﺞ ﺍﻭﻀﺤﺕ ﺍﻥ

ﺍﻟﻌﺯﻻﺕ ﻜﺎﻨﺕ ﺘﺘﺸﺎﺒﺔ ﻓﻲ ﺼﺒﻐﺔ ﺠﺭﺍﻡ ﺠﻤﻴﻌﻬﺎ ﺴﺎﻟﺒﺔ ﻟﺼﺒﻐﺔ ﺍﻟﺠﺭﺍﻡ ﻭ ﻜﻠﻬﺎ ﺘﻨﺘﻤﻲ ﺍﻟﻲ ﻋﺎﺌﻠﺔ

ﺍﻟﻤﻌﻭﻴﺎﺕ ﺍﻻ ﻋﺯﻟﺔ ﻭﺍﺤﺩﺓ.

ﺍﺨﺘﺒﺎﺭﺍﺕ ﺍﻟﻜﻴﻤﻴﺎﺀ ﺍﻟﺤﻴﻭﻴﺔ ﺃﻭﻀﺤﺕ ﺍﻥ ﺴﺕ ﻭﺜﻼﺜﻭﻥ ﻋﺯﻟﺔ ﺘﻨﺘﻤﻲ ﺍﻟﻰ ﺒﺎﻜﺘﻴﺭﻴﺎ

ﺍﻻﺸﻴﺭﻜﻴﺔ ﺍﻟﻘﻭﻟﻭﻨﻴﺔ ﻫﺫﺍ ﻴﺸﻴﺭ ﺍﻟﻰ ﺍﻨﻬﺎ ﺍﻻﻜﺜﺭ ﺇ ﻨ ﺘ ﺸ ﺎ ﺭ ﺍﹰ ﺤﻴﺙ ﺃﻨﻬﺎ ﺘﻤﺜل (٧٠,٥% ) ﻤﻥ ﺠﻤﻠﺔ ﺍﻟﺒﻜﺘﺭﻴﺎ

ﺍﻟﻤﺼﺎﺤﺒﺔ ﻹﺴﻬﺎﻻﺕ ﺍﻷﻁﻔﺎل ﻭ ﺍﻴﻀﺎ ﺘﻡ ﻋﺯل ٩ (١٧,٧%)ﺍﻟﺴﺭﺍﺘﻴﺔﺍﻟﺒﻭﻟﻴﻤﺜﻜﺔ ﻭ٩ (١٧,٧%)

ﺍﻻﻨﺘﺭﻭﺒﺎﻜﺘﻴﺭ ﻜﻭﻟﻭﺍﻜﻲ ﻭ ٧ (١٣,٧%) ﺍﻟﻜﻠﺒﺴﻴﺔ ﺍﻟﺭﺌﻭﻴﺔ ﻭ٥(٩,٨%) ﺍﻟﻤﺘﻘﻠﺒﺔ ﺍﻻﻋﺘﻴﺎﺩﻴﺔ ﻭ٤ ( ٧,٨%) ﺍﻟﺴﺭﺍﺘﻴﺔ ﺍﻟﺯﺍﺒﻠﺔ ﻭ ٣ (٥,٩%) ﺍﻟﻤﺘﻘﻠﺒﺔ ﺍﻟﺭﺍﺌﻌﺔ ﻭ ٣ (٥,٩%) ﺍﻟﺴﻴﺘﺭﻭﺒﺎﻜﺘﺭﻓﺭﻴﻨﺩﺍﻱ

ﻭ٣(٥,٩%) ﺒﺭﻭﻓﻴﺩﻨﺴﻴﺎ ﺍﻟﻜﺎﻟﺒﻔﻴﺸﻴﻨﺕ ﻭ ﻋﺘﺭﺓ ﻭﺍﺤﺩﺓ (٢%) ﻟﻜل ﻤﻥ ﺍﻟﺸﻴﻘﻴﻠﺔ ﺍﻟﺯﺤﺎﺭﻴﺔ ﻭﺍﻟﻴﺭﺴﻨﻴﺔ

ﺍﻟﻭﺴﻁﻴﺔ ﻭﺍﻟﺴﻠﻤﻭﻨﻴﻠﺔ IV ﻭﺍﻟﺴﻠﻤﻭﻨﻴﻠﺔ III. ﺒﺎﻹﻀﺎﻓﺔ ﺍﻟﻲ ﻋﺎﺌﻠﺔ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻤﻌﻭﻴﺔ ﺘﻡ ﺍﻟﺤﺼﻭل ﻋﻠﻲ

ﻋﺯﻟﺔ ﻭﺍﺤﺩﺓ ٢% ﻤﻥ ﺍﻟﺯﺍﺌﻔﺔ ﺍﻟﺯﻨﺠﺎﺭﻴﺔ.

ﺍﺨﺘﺒﺭﺕ ﻓﻌﺎﻟﻴﺔ ﺴﺕ ﻤﻥ ﺍﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﺤﻴﻭﻴﺔ ﻭ ﻤﺴﺘﺨﻠﺼﺎﺕ ﻟﻨﺒﺎﺘﻴﻥ ﻁﺒﻴﻴﻥ ﻋﻠﻲ ﺴﺒﻊ ﻭﺴﺘﻭﻥ

ﺠﺭﺜﻭﻤﺔ ﻋﺯﻟﺕ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻁﺭﻴﻘﺔ ﺍﻟﺘﺨﺭﻴﻡ.

ﺃﻭﻀﺤﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺍﻥ ﺍﻟﺯﻴﺕ ﺍﻟﻁﻴﺎﺭ ﻟﻨﺒﺎﺕ ﺍﻟﻴﻨﺴﻭﻥ ﺒﺘﺭﻜﻴﺯ ١٠% ﺜﺒﻁ ﻨﻤﻭ ﺍﺭﺒﻊ ﻭﺴﺘﻭﻥ

(٩٥,٥%) ﻋﺯﻟﺔ ﺍﻤﺎ ﺍﻟﺘﺭﺍﻜﻴﺯ ١% ﻭ٠,١% ﺜﺒﻁﺘﺎ ﻨﻤﻭ ٦٣(٩٤%) ﻭ ٥٧(%٨٥,١) ﻤﻥ ﺠﻤﻠﺔ

ﺍﻟﻌﺯﻻﺕ ﺍﻟﺘﻲ ﺃﺨﺘﺒﺭﺕ ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ ، ﻜل ﺍﻟﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻟﻤﺎﺌﻴﺔ ﻟﻨﺒﺎﺕ ﺍﻟﺘﺭﺘﻭﺱ ﻟﻡ ﺘﺜﺒﻁ ﺍﻟﻨﻤﻭ ﻷﻱ ﻤﻥ

ﺍﻟﻌﺯﻻﺕ ﺍﻟﺘﻲ ﺃﺨﺘﺒﺭﺕ.

ﺍﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﺤﻴﻭﻴﺔ ﺘﺘﺒﺎﻴﻨﺕ ﻓﻲ ﻤﻘﺩﺭﺘﻬﺎ ﻋﻠﻰ ﺘﺜﺒﻴﻁ ﻨﻤﻭﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ. ﺍﻜﺜﺭ ﺍﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﺤﻴﻭﻴﺔ

ﻓﻌﺎﻟﻴﺔﻜﺎﻥ ﺍﻟﻨﺎﻟﻴﺩﻜﺴﻴﻙ ﺍﺴﺩ ﺤﻴﺙ ﺜﺒﻁ ﻨﻤﻭ٩٤% ﻤﻥ ﺠﻤﻠﺔ ﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ ﺍﻟﺘﻲ ﺍﺨﺘﺒﺭﺕ ﻭﺍﻟﺠﻨﺘﺎﻤﺎﻴﺴﻴﻥ ﺜﺎﻨﻲ

ﺍﻟﻤﻀﺎﺩﺍﺕ ﻓﻌﺎﻟﻴﺔ ﺤﻴﺙ ﺜﺒﻁ ﻨﻤﻭ٩٢,٥% .ﺃﻤﺎ ﺍﻟﺒﻨﺴﻠﻴﻥ ﻜﺎﻥ ﺃﻗل ﺍﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﺠﺭﺜﻭﻤﻴﺔ ﺘ ﺄ ﺜ ﻴ ﺭ ﺍﹰ ﻭﺜﺒﻁ ﻨﻤﻭ

٩(١٣,٥ %) ﻤﻥ ﺍﻟﺠﺭﺍﺜﻴﻡ ﺍﻟﺘﻲ ﺍﺨﺘﺒﺭﺕ.

INTRODUCTION

Diarrhoea is loosely defined, as passage of abnormally liquid or uniformed stool at an increase frequency, for adult on a typical westren diet g per day can generally be considered as ٢٠٠ stool weight more than weeks and ٢ diarrhoeal. Diarrhoea may be further defined as acute if less than ,weeks in duration (Kasper ٤ weeks and chronic if more than ٤-٢ persistent if .(٢٠٠٥ Diarrhora is a common manfistigation of a complex of diseases which affect human. Diarrhoea also is one the major causes of morbidity and mortality among preschool children in developing countries. Acute bacterial diarrhoeal disease is a worldwide problem of enormous magnitude. Diarrhoeal diseases are one of leading causes of morbidity and mortality in children in worldwide, causing one billion episodes of illness and Mortality due to .(٢٠٠٤ ,.million death annually (Behrman et al ٥-٣ diarrhoeal diseases in children below five years was identified as a largest In Sudan the mortality rate among .(١٩٧٩ ,single cause of mortality (Mahler and most of this percentage was due to diarrhoeal ٪٩ children estimated as In Sudan diarrhoeal disease in children is one of .(٢٠٠٤ ,disease (Abdagader death of children below five years occur per year ٢٠٠٠ big problems, about .(١٩٨١ ,Taha) The bacteria isolated from faecal samples of infants with diarrhoea exhibit high drug resistance to antimicrobial in common use in .(٢٠٠٥ ,Khartoum state (Mohammed Sudan is one of the countries that commonly used folkloric medicine because it has many types of medicinal plants. Diarrhoea is one of the major disease that controlled by folkloric medicine. Pimpinella anisum and Hydnora abissynica are used for treatment of child diarrhoea so this study is intended to isolate bacteria associaed with child diarrhoea in Omdurman area,to examine antibacterial effect of P.anisum andH.abissynica on the diarrhoeal bacteria isolates and also to compare antibacterial activity of these medicinal plants with antibiotics in common use for treatment of child diarrhoea The aim of this study: .To isolate and identify bacteria that causes child diarrhoea (١) To examine antibacterial activity of antibiotic used for treatment child (٢) diarrhoea in the Omdurman area on bacterial isolates associated with child diarrhoea. To test sensitivity of isolates to anise Pimpinella anisum and Hydnora (٣) abissynica. To compare the antibacterial activity of medicinal plants with antibacterial (٤) activity of antibiotics.

CHAPTER ONE LETRETUER REVIEW .١ :Diarrhoea ١٫١ :Definition ١٫١٫٢ The term of diarrhoeal disease is loose wide term; it has different term in this field different author speak of infantile diarrhoea, gastroenteritis, .(١٩٦٤ ,summer dirrhoea and even dysentery (Wison and Mile Diarrhoea is loosely defined as passage of abnormally liquid or uniformed stool at an increase frequency, for adult on a typical westrent diet .g/day can generally be considered diarrhoeal ٢٠٠ stool weight exeed Diarrhoea may be further defined as acute if less than two weeks and it weeks and chronic if more than four weeks in duration ٤-٢ persistent if .(٢٠٠٥ ,Kasper) ,h ٢٤/g ٢٥٠ Diarrhoea is defined as a stool weight of more than but quantification of stool is necessary only in some patient with chronic diarrhroea. In some cases the physician's working definition of diarrhoea increase stool frequency (more than two or three bowel movement per day) or .(٢٠٠٤ ,.liquidity of faeces (Tierney et al defined diarrhoea, as increased in total (٢٠٠٤) .Behrman et al daily stool output which is usually associated with stool water content. For .h ٢٤/g/kg ١٠ infants and children this would result in stool output greater than .weeks is considered as chronic ٢ Diarrhoea lasting longer than :Classification ١٫١٫٣ :Acute diarrhoea ١٫١٫٣٫١ weeks ٣ Diarrhoea of acute onset and persisting not more than commonly caused by infectious agents, bacteria toxin or drugs (Tienery et al., of cases of diarrhoea are caused by infectious agent the ٪٩٠ More than .(٢٠٠٤ or so are caused by medications, toxic ingestion ischemia and ٪١٠ remaining .(٢٠٠٥ ,other conditions (Kasper Acute diarrhoea usually due to faecal – oral transmition of bacterial toxin, viruses, bacteria or protozoan organism, it has short – lived (Kasper, .(٢٠٠٥ stated that acute diarrhoea can be divided into (٢٠٠٤) .Tiernery et al noninflammatory and inflammatory. The noninflammatory diarrhoea is watery, nonbloody diarrhoea caused by either toxic producing bacterium enterotoxogenic E. coli (ETEC) or other agent such as viruses that disrupt normal absorption and secretory process in the small intestine. Inflammatory diarrhoea characterized by the presence of fever and bloody diarrhoea which indicates colonic tissue damage caused by invasion ( or yersinia .(H٧ :infection) or a toxin (E. coli O١٥٧ Inflammatory diarrhoea occurs because of damage to intestinal mucosal cells that there is a loss of fluid and blood also there is defective absorption of .(٢٠٠٢ ,fluid and electrolyte (Kumar and Clark :Chronic diarrhoea ١٫١٫٣٫٢ The most common cause of chronic diarrhoea irritable bowel syndrome rarely occurs at night and most severe before or after breakfast. The stool g/day, it ٢٠٠ contain mucus but never blood and volume of stool is less than can be categorized as disease of the colon or small bowel or malabsobtion, .days ١٤ also chronic diarrhoea define as a diarrhoea persisting for more than Enteropathogenic E. coli (EPEC) is only bacteria that cause chronic diarrhoea .(٢٠٠٢ ,.Haslett et al) :١٫١٫٤Causative agent of diarrhoea Diarrhoea caused by various causative agents such as bacteria, virus, parasite and other causes: a. Bacteria: Aeromonas, Bacillus cereus, Campylobacter jejnni, Shigella Vibrio ,and O١٣٩ spp, Staphylococcus areus, O١ parahaemolyticus, and Eschericha coli (Behrman et (٢٠٠٤ ,.al b. Virus: Rotavirus, Astrovirys, Calicivrus, Noroviruses, Cytomegalovirus . .(٢٠٠٤ ,.and Herbes simlex viruses (Behrman et al c. Parasite: Strogyloidis stercoralris, Balantidium coli, Blastocytis hominis, Cryptospridium parvum, Cyclospora cayetanensis, Isospora belli, Giardia lamblia, Trichuris trichiura, Entamoeba hitolytica and others. .(٢٠٠٤ ,.Behrman et al) d. Other causes: Anatomic difficulty (e.g malrotation, faecal impaction, bowel short), malabsobtion ( e.g disaccharide deficiencies, glucose-glactose malabsobtion),endocrinopathies (e.g thyrotoxicosis, Addison disease, adrenogental syndrome), food poisoning(e.g heavy metal scombroid)) and neoplasm (e.g neuroblastomas, carcinoid and Zollinger-Ellison syndrome) and miscellaneous ( e.g milk allergy, pellagra, laxative abuse, Crohn disease ,ultration colitis, acrodermatitis, immunodeficiency disease and others) .(٢٠٠٤ ,.Behrman et al) :Mechanisms ١٫١٫٥ Bacteria can cause diarrhoea in three different ways. Some species may employ more than one of these methods: :Mucosal adherence ١٫١٫٥٫١ Bacteria adhere to specific receptor on the mucosa, there are many adhesions molecules protrude from bacterial surface aid adhesion (e.g. Escherichia coli EPEC) damage the mucosa and produce a secretary diarrhoea .(٢٠٠٢ ,directly as a result of adherence (Kumar and Clark :Mucosal invasion .١٫١٫٥٫٢ Invasion by such as Shigella spp, campylobacter spp and Enteroinvasive E. coli (EIEC) penetrate into the intestinal mucosa, they destroy the epithelial cell and produce the symptoms of dysentery, some Gram negative bacteria actually transport molecules e.g. toxin from their cytoplasm to the outside these system vary from simple protein complex on the bacterial surfaces to highly complex mechanisms where protudimg hollow needle is formed to inject toxins into the host cell ( e.g. Yerisinia, Salmonella) the injected protein have been shown to homology with host cells kinase and phospholylase and thus can modulate the host snalling (e.g atyrosine phosphate) which causes cytoskeletal rearrangments and reduces the rate of phagocytosis in infected macrophages allowing continued multiplication .(٢٠٠٢ ,Kumar and Clark) :Toxin production .١٫١٫٥٫٣ Diarrhoea can be caused by three different types of bacterial toxin: a) Enterotoxin: Induce excessive fluid into the bowel lumen, leading to watery diarrhoea, without physical damaging the mucosa, e.g. Salmonella spp, .(٢٠٠٢ ,Enterotoxogenic E. coli (ETEC) (Kumar and Clark b) Neurotoxin: Affect the autonomic nervous system causing diarrhoea and vomiting .(٢٠٠٢ ,e.g. Bacillus cereus(Kumar and Clark c) Cytotoxins: Damage the intestinal mucosa and, in some cases vascular endothelium cells and usually these toxins are produced by bacteria adhering to the intestinal epithelium e.g. Salmonella spp , campylobacer spp and .(٢٠٠٢ ,Enterohaemorrhagic E. coli (EHEC) (Kumar and Clark :Magnitude of the problem ١٫١٫٦ Mortility due to dairrhoeal disease in children below five years is Diarrhoea as .(١٩٧٩ ,identified as largest single cause of mortility (Mahler such is considered as a top of killer of children below five year of age by .(١٩٨٤ ) WHO Acute gastroenteritis represents a frequent cause of morbility and mortility among children in developing world as well as morbidity, in .(١٩٩٩ ,developed world (Gastanduy and Beque Infant mortily rate is unknown in most tropical countries but most .(١٩٨٢ ,death per thousand (Oklashialsm ٣٠٠-١٠٠ estimation between Diarrhoeal disease is identified as a largest single cause of death among Diarrhoea diseases are one of .(١٩٧٣ ,children in developing world (WHO leading cause of morbidity and mortality of children worldwide, causing one ,.million deaths annually (Behrman et al ٥-٣ billion episodes of illness and .(٢٠٠٤ Gastroenteritis and diarrhoea are major cause of death in many developing countries especially inadequate water supplies and areas of poor .(١٩٨٩ ,sanitation and little or no health education (Cheesbrough :Transmission and spread of infection ١٫١٫٧ Food and drink of children are main way of infection. These contaminations happen by various ways either before or after preparation of meal. Poor personal and environmental hygiene of the mother or individual nursing of child is great of importance, infection may result directly from mother to child e.g. Escherichia coli and shigella infection. The bad care feeding bottles and other utensils of food and drink of child also factors of transmission. Water is usually contaminated by sewage or direct defaecation .(١٩٧٥ ,in it or in irrigation cannel (Erwa Most people store water in large containers known as (Zeer) which cool water. A mug is container near (Zeer) for user by every body in the house, the mug filled by immersing it in the Zeer and most fingers touch the water and However, in general, water.(١٩٦١ ,lead to contamination (WHO contamination is more importance in salmonella and vibrio infections. Shigella infection is often a result of food or milk contamination. E. coli infection may occur under all circumstance, objectives such as door knobs, children's toys and pumpus, toilet seats ….ect may transmit infection. The carriers of infection are usually mothers, nurses, and often children who may be ill or only healthy carriers. Flies (house fly or Musca domestica) are one of importance role of transmission of infection. Flies act as a mechanical carrier. Qutaishat et al. .reported that transmission of Salmonella entirica serotype S (٢٠٠٣) to infant via the mothers breast (DT١٠٤) ١٠٤ typhimurium defective type milk. The major mechanisms of transmission for diarrhoeal pathogens are person to person through feacal oral route or by ingestion of contaminated (٢٠٠٥ ,food or water (Mohammed .two outbreaks of Salmonella infection originated in Victoria ١٩٧٠ In One of them by contamination of infant powdered milk formula during the manufacturing and the second outbreak via carries, working in sandwich .(٢٠٠٣ ,.Forsyth et al) :Diarrhoea in Sudan ١٫١٫٨ reported a significant incidence of diarrhoea disease and (١٩٧٥) Erwa gastroenteritist in neonates. The study was carried out in Khartoum North and showed a higher prevalence of Enteropathogenic Escherichia coli (EPEC). of ٪٤٦ revealed that (١٩٧٧) Data from Khartoum emergency hospital causes death in children who were hospitalized in that year was due to of ٪٣٧٫٨ gastroenteritis. Malaria was found to be cause diarrhoeal disease, as cases of diarrhoea were accompanying an attack of malaria in children below .(١٩٨٥ ,.the age of five at Juba town (Sharaf et al reported that the high incidence of acute gastroenteritis (١٩٨٦) Sharaf occur during the summer and in autumn, also he mention that diarrhoea is the major causes of morbidity and mortality in children especially in the acute stage or when attack are repeat. Diarrhoeal disease is the most important cause of the death in Sudan (١٩٧٣ ,Annual Report) Diarrhoea is one of the major causes of deaths amongst infants the world over; and especially widespread in less development societies, the poor personal and community hygiene practice, bad sanitation, unhealthy water supply source and neglect of hygiene altogether contribute to the spread of .(٢٠٠٥ ,infection (Mohammed :Enterobacteriaceae ١٫٢ Enterobacteriaceae are facultative anaerobic Gram – negative bacteria that can be motile or non motile, the motile strains have peritrichous flagella, all species are grow well on artificial media and attack glucose, can not reduce nitrate except genus Erwina which reduce nitrates to nitirites. The antigenic composition contain a mosaic of interleukin relationship among the several genera.They can be divided into lactose - fermenting and non lactose – fermenting group, the lactose – fermenters include Encetrobacter, Echerichia and Klebseilla. Also some Salmonella can ferment lactose (Koneman et al., .(١٩٩٧ :Taxonomy and classification of Enterobacteriaceae ١٫٢٫١ species, Farmer ٢٦ genera and ١١ ,described (١٩٧٢) Edward and Ewing enteric ٢٩ species and ٦٩ genera composed of ٢٢ described (١٩٨٥) .et al species, biogroup and ١٣٩ ,genera ٣١ group.Enterobacteriaceae contain .(١٩٩٧,.unnamed enteric groups (Koneman et al :reported that Enterobacteriaceae include (١٩٩٢) .Koneman et al .Escherichiae, this includes Escherichia and Shigella :١ Trribe Tribe II: Edwardsielleae, this includes Edwardsiella species. Tribe III: Salmonella. Tribe IV: Citrobactereae. Tribe V: Klebsilleae, this includes Klebsiella, Enterobacter Hafnia, Pantoea and Serratia species. Tribe VI: Proteae, this includes, Proteus, Morgenella and Providencia species. Tribe VII: Yersinieae include Yersinia species. :Escherichia ١٫٢٫٣ Strains of Escherichia coli are related to Gram – negative ‘coliform’ found in the gut of man and animal. They are usually motile some may produce polysaccharide capsule. They grow well on non selective media, most strains ferment lactose producing large red colonies in MacConkey's agar. E. coli can be differentiated from other member of Enterobacteriaceae by ability to utilize sugar and biochemical reactions, such as indole production and formation of acid and gas from lactose and other carbohydrate. Ecsherichia .(٢٠٠٢ ,.C (Greenwood et al°٣٧ C as well as°٤٤ take place at :Pathogenisis of E. coli ١ ١٫٢٫٣ E. coli besides being a normal inhabitant of intestinal tract, E.coli is also associated with variety pathological condition including gastroenteritis in .(٢٠٠٢ ,.Greenwood et al ;١٩٧٠ ,man and animal (Sojka E. coli also found in gut of bird, reptile and amphibians, E.coli is excreted in a great numbers with faeces and found external environment. It is opportunistic organism that causes wide and variety of infections in several part of the human and animal body where some abnormalities or impairment of host defense. The virulence factors of E. coli are present on fimbriae ,and Levine ١٩٨٣ ,.adhesion) and production of entrotoxins (Levine et al) .(١٩٨٧ E. coli strains that cause diarrhoea have been recently classified into six .(١٩٩٨ and Nataro and Kaper ٢٠٠٠ ,.groups (Okeke et al According to their mechanism of pathogenesis and their corresponding clinical symptoms in man E. coli are classified as follow. (Enterotoxogenic E. coli (ETEC ١٫٢٫٣٫١٫١ These strains cause acute watery diarrhoea, sometime of -like severity particularly in developing country. It is associated with diarrhoeagenic children under five year of age. ETEC colonized small intestine and produce one or both enterotoxins heat labile (HL) cholera –like toxin and heat stable (HS) toxin .They also contributed a common of worldwide diarrhoea in people travlling from temperate areas with good .(١٩٩٣ ,hygiene to mainly warm countries with poor hygiene (Gyles Enterotoxigenic Ecsherichia coli (ETEC) usually express fimbraie that are specific for the host animal species and which enable the organism to adhere to epithelium of the small intestine in addition to production of one or both toxins. Enterotoxin done not sufficient to cause diarrhoea. The organism must initially to be able to adhere to the mucosal surface of epithelial cells of small intestine. This adhesion mainly mediated by fimbriae that bind to specific receptors in the small intestine. These adhesions fimbriae are termed colonization factor antigens (CFAs). reported that ETEC are major cause of (٢٠٠٢) .Greenwood et al years in developing countries, and in ٥ mortality in children under the age of travelers visiting countries where ETEC are endemic. The source and mode of spread of ETEC infection in warm – climate countries are not well understood, but it seem likely that water contaminated by human and animal sewage plays an important part in the spread of infection. ETEC is a major cause of infantile diarrhoea in developing countries as well as important etiologic agent of traveler’s diarrhoea. They account about .(٢٠٠٤ ,of diarrhoeal episodes in developing countries (Behrman ٪٣٠-٢٠ ETEC is a major cause of endemic diarrhoea in tropical and developing -٢٥ countries and most common agent of traveler’s diarrhoea causing about (١-of cases. Disease is mediated primarily by heat – labile toxin (LT ٪٧٥ and/or a heat – stable toxin (STa) that causes net fluid secretion via activation and/or (STa) guanylate cyclase activated in (١-of adenylate cyclase (LT jejunum and ileum; this result is watery diarrhoea accompanied by cramps. consists of an A and B subunit and is structurally and functionally ١-LT similar to cholera toxin. ETEC produce both heat – stable and heat labile enterotoxin, these toxin stimulate secretion of fluid into the intestinal lumen. Transmission is normally via contaminated food. The organism is a major cause of traveller’s diarrhoea .(٢٠٠٢ ,Kumar and Clark) :(Enteroinvasive E. coli (EIEC .١٫٢٫٣٫١٫٢ In developing countries sporadic disease is frequently recognized in children and travelers by EIEC. It shares many genetic and clinical features with shigella, but it cause disease only by large number of inoculums. Incubation period about one to three days. Initially, enterotoxins are believed to induce secretory small bowel diarrhoea, subsequently colonization and invasion of colonic mucosa, followed by replication them inside the cells and cell – to – cell spread, result in development of inflammatory colitis characterized by fever, abdominal pain and scant stool with mucus, blood and inflammatory cells symptoms are usually self limited .(٢٠٠٥ ,.Kasper et al) of sporadic diarrhoea ٪٥ reported that about (٢٠٠٤) .Behrman et al of bloody diarrhoea may be caused by EIEC. Inflammatory ٪٢٠ episodes and .and others (H٧:٠١٥٧ diarrhoea is associated with EIEC (E. coli EIEC invades the colonic mucosa, producing widespread mucosal .(٢٠٠٢ ,damage with acute inflammation (Behrman and Kuegman EIEC and Shigellae cause disease by invading intestinal epithelial. Infection caused mainly by ingestion. Ingestion of small number of bacteria is needed to produce infection, they are relatively resistant to gastric acid and bile, and pass readily into the large intestine, where they multiply in the gut lumen. The bacteria overlay mucus layers, attach to the intestinal epithelial cells and carried into the cell by endocytosis into endocytic vacuoles which then lyses. The ability to cause vacuoles to lyses is an important virulence attributes,as organisims unable to do this can not spread to the neighboring cells. After lyses of the vacuoles the bacteria multiply within the epithelial cells and kill it, spread to neighboring cells lead to tissue destruction and consequent inflammation. Pathogenicity of Shigella and EIEC depend on both chromosomal and plasmid genes. A large plasmid carried genes for expression of outer membrane protein that are required for invasion as well as genes that may be necessary for the insertion of this protein into the cell membrane. Plasmid genes are also required for ability to escape from endocytic vacuole and to invade contagious host cells, chromosomal genes encoding pathogenic mechanisms include those required for expression of large chain LPS and those encoding aerobactin iron – sequestering system (Greenwood et al., .(٢٠٠٢ :(Enteropathogenic E. coli (EPEC ١٫٢٫٣٫١٫٣ EPEC cause disease primarily in young children, including neonates. The first E. coli pathotype recognized as an agent of diarrhoeal diseases. EPEC was responsible for outbreak of infantile diarrhoea. In developing countries infection of enteritis is still common in the communities with poor hygiene, in these countries sporadic cases and outbreaks occur very frequently .(٢٠٠٢ ,.in the general community as well as in institutions (Greenwood et al Breast – feeding diminishes the incidence of EPEC infection, rapid days). Initial ٢-١) person – to – person spread may occur. Incubation period localized adherence lead to characteristic effacement of microvilli with formation of cup like action – rich pedestals. Diarrhoeaal stool often contain ,(days ١٥-٥ mucus but not blood although self – limited may ocur (lasting for .(٢٠٠٥ ,.EPEC diarrhoea may be persistent for several weeks (Kasper et al EPEC has ability to produce a characteristic alteration in the microvillus membrane, the “attaching and effacing” lesions. They are major cause of infant diarrhoea, mainly in developing countries primarily in children less than .(٢٠٠٤ ,.years (Behrman et al ٢ EPEC cause diarrhoeal disease in infants in developing countries. They express verocytotoxin colonization of the upper part of small intestine occur in infantile enteritis associated with EPEC. Adhesion and mucosal damage has been termed in cattaching and effacing lesions. The organism cause this phenotpe has been termed attaching and effacing E. coli (AEEC). The genes responsible are located on locus for enterocyte effacement pathogenicity island located on the E. coli chromosomes. One of the proteins involve, intimin appear to be key EPEC adhesin. These EPEC also synthesize a translocated intimin receptor, which is inserted into the host gut wall providing a binding site for intimin. These strains of EPEC use a novel mechanism of adhesion where both adhesion and receptor are synthesized by .(٢٠٠٢ ,.bacteria(Greenwood et al :(Verocytotoxic E. coli (VTEC ١٫٢٫٣٫١٫٤ Strains of E. coli expressing a protein cytotoxic for verocells were These bacteria became important because it linked with .١٩٧٧ discovered in two disease previously of unknown etiology, haemorrohagic uraemic syndrome and haemorrhagic colitis. The ability to cause haemorrhagic colitis has led some workers to refer to these strains as enterohaemorrohagic E. coli (EHEC). The biological properties, physical characteristics and antigenicity of verotoxic(VT) are very similar to those of shiga toxin. Serological tests have Antibodies prepared .and VT٢ revealed two antigenically distance forms VT١ -do not. Shiga neutrilize shiga toxin, while antibodies specific for VT٢ to VT١ comprised A and B subunits. The A subunit and VT٢ like toxin, VT١ possesses the biological activity of toxin while the B subunits mediate specific bind to and VT٢ binding and receptor – mediated uptake. VT١ molecules present on the surface of certain (globotriosylceramide (Gb٣ variant toxin binds to globotertraosylcermide eukaryotic cells. In contrast VT٢ During infection with VTEC the inflammatory mediators is tumor .(Gb٤) necrosis factor (TNF) and interlecukin- I (IL-I), in combination with LPS, increase the number of cermide receptor on the surface of eucharistic cells, enhancing the binding of VT to these cells. Infection also result in expression receptor located in the kidneys leading to of VT molecules that bind to Gb٣ haemolytic uraemic syndrome, VT once bound to eukarytic cell surface, the holotoxin becomes internalized by host cells and remain active within endosome. The toxin eventually reaches the golgi apparatus by mechanisms as yet unknown. A subunit becomes enzymatically (lniked) to form portion A١ portion of toxin prevents protein synthesis and cell The A١ .and A٢ . (٢٠٠٢ ,.death(Greenwood et al :(Entero – aggregative E. coli (EAggEC ١٫٢٫٣٫١٫٥ EAggE have ability to adhere to particular laboratory – culture cell such in aggregative or stacked brick pattern. Strains of EAggEC were ٢ – as HEp as a cause of chronic diarrhoea in malnourished young ١٩٨٧ first reported in may be putative pathogenic ٢ -children living in Chile. Adhesion to HEp ٢ –mechanism also strain of EAggEC may express fimbriae. Adhesion to HEp cells can occur in absence of these structures and for certain strains adhesion involves cell – surface charge. The site of adhesion in the human host has been not determined. Strain have been reported to produce an ST – like toxin, but this has not been demonstrated in all EAggEC and mechanism by which these strains cause diarrhoeal disease illness are poorly understood, some isolates express haemolysin, an aerobactin – mediated high – affinity iron uptake system and a range of haemogglutinin; however, a part from ability to adhere cells in the stacked – brick formation, these strains are generally ٢ -to HEp .(٢٠٠٢ ,.quite distinct (Greenwood et al In EAggEc a symptomic excretion rate can be high. It is associated with days) pediatric diarrhoea in developing countries most ١٤ < persistent (lasting EAggEC .(٢٠٠٤ ,.month of age (Behrman et al ١٢ < prominently in children are etiologic agent in AIDS – associated chronic diarrhoea and traveler’s reported that (١٩٩٨) Nataro and Kaper .(١٩٩٢ ,.diarrhoea (Bandry et al EAggE cause persistent diarrhoea. have reported that non haemolytic EAggE strain (١٩٩٢) .Beldwin et al produce a heat – labile toxin which antigenically related to E. coli haemolysin. :(Diffuse adherent E. coli (DAEC ١٫٢٫٣٫١٫٦ DAEC strain has been described primarily in developing countries and in young children, this strain can cause traveler’s diarrhoea. Large inoculum is required for infection, in vitro the organism exhibit a diffuse or “stacked - brick” adherence pattern. Clinical disease has been associated with prolonged .(٢٠٠٤ ,.watery diarrhoea (Behrman et al :Viability of E. coli ١٫٢٫٣٫٢ minutes though ٣٠ C for°٦٠ E. coli is killed by moist heat at minutes, survive ٣٠ C for° ٦٣-٦٢ occasionally some survive pasteurization at in water for several week or months. It grow in natural water outside of the body, it also can survive several days when dried in clothing or dusts. Pathogenic serotype may be viable and numerous in floor dust, in air and .(١٩٨٩ ,equipment of hospitals (Sleigh and Duguit Some strains are more heat resistant than other members of ٦٠ C for°٥٥ minutes or ١٥ C for°٦٠ Enterobacteriaceae and may survive C (Greenwood°٤٥-١٥ minutes. E. coli grows over wide range of temperature .(٢٠٠٢ ,.et al :Shigella species ١٫٢٫٤ :Description ١٫٢٫٤٫١ They are generally non lactose fermenters and biochemicaly inert, and do not produce gas from carbohydrates except certain biotypes of Shigella flexineri are aerogenic, rare strain biotype of S. sonnei can strogly ferment lactose and sucrose and most strain of Shigella can decarboxylate ornithine. :Classification ١٫٢٫٤٫٢ The classification of Central Disease Control (CDC) Compose of: S. dysenteriae (Group A) S. flexeneri (Group B) S. boydii (Group C) S. sonnii ( biochemically different from other Shigella species). Shigellae are Gram negative non motile rods, selective media such as SS agar, XLD and DCA are required to isolate Shigella spp from faeces. On (١٩٨٩ ,SS agar it produces non lactose fermenting colonies (Geesbrough Antimicrobial drug with activity against Shigella species include Sulphanamine, Teteracycine, Chloramphenicol, Ampicillin and Sterptomycin. In recent years strains of Shigellae resistant to sulphonamides and ,Infants below six months.(١٩٨٩ ,streptomycin is reported (Geesbrough especially neonates are rarely infected with Shigellae in contrast to .(١٩٩٧ ,.Salmonellae (Koneman et al :١٫٢٫٤٫٣Incidence and source of shigella infection Shigellosis is most communicable bacteria disease that cause diarrhoeas. Human serve as the nature host, the disease is transmitted by fecal ,viable organisms being able to cause disease ٢٠٠ oral route with as few as – Shigella sonei is almost serotype commonly associated with diarrhoeal disease .(١٩٩٧ ,.Koneman et al) :Pathogenesis .١٫٢٫٤٫٤ The basic virulence factor shared by all Shigella spp has ability to invade intestine. This charactaristic encode large plasmid that responsible for synthesis of group of polypeptides involved in cell invasion and killing. Chromosomally encoded factor are also required for full virulence, some of these chromosomal traits are important for all Shigellae (e.g. lipopolyscharide synthesis). Shiga toxin also important factor for others ( Shiga toxin is potent protein synthesis interference , inhibiting .(١ serotype ١ exotoxin produced insignificant amount by Shigella dysenteriae serotype .Watery diarrhoea of shigellosis may be caused by enterotoxin, The pathogenic change primary take place in colon. The changes are most intense in the distal colon, although pancolitis may occur. Grossly, localized and diffuse mucosal odema, ulceration, friable mucosa, bleeding and exudates may be seen. Microscopically ulcerations, epithelial cells death and pseudomembrane, infiltratin extended from the mucosa to mucscularis mucosae by polymorphonuear cells and mononuclear cells and submucosal .(٢٠٠٢ ,.edema occur (Greenwood et al :Proteus species .١٫٢٫٥ :Normal habitat ١٫٢٫٥٫١ Proteus species are found in intestine of human and animals, soil, added (١٩٧٧) Buxton and Fraser .(١٩٨٩ ,sewage and water (Cheesbrough Proteus spp may be associated with otitis and peritonitis. Sleigh and Duguit reported that they are associated with urinary tract infection and wound (١٩٨٩) infection. :General characteristic ١٫٢٫٥٫٢ Proteus mirabilis and P.vulgaris are active motile, non- -٣٥ capsulated,Gram – negative pleomorphic rod, motility is not observed at C, but motile at room temperature. Most strains produce characteristic°٣٧ swarming growth when cultivated aerobically over surface of blood agar and several other media but inhibited on media contained bile salts such as MacConky agar and Salmonella-Shigella (SS)agar, individual non lactose ,C°٣٧-٣٥ fermenting colonies are produced after overnight incubation at The .(١٩٩٤ ,Holt et al ;٢٠٠٠ ,Proteus spp have distinctive smell (Cheesbrough .(١٩٩٤ ,organisms are methyl-red negative and VP negative (Holt et al stated that these organisms may cause abdominal and (١٩٨٩) Cheesbrough wound infections also secondary invader to ulcer pressure and damaged tissue. The organism may also cause septicemia meningitis chest infections. P. vulgaris isolated occasionally from urine, pus and other specimen. P. mirabilis infections usually responded to antimicrobial thereby than those cause by P. vulgaris. Many strains of two species of proteus produce bacteriocin which has lethal action against other organism. A new method of typing of strains by determination of their paticin, the method was more discriminated when it was .(١٩٧٧ ,used in combination with O serotyping (Senior :Antibiotic sensitivity .١٫٢٫٥٫٣ Antibiotic with activity against P. mirabilis include Ampicillin, Cephalosporin and Aminoglycosides. Some strains of P. mirabilis are beta lactamase producing and therefore resistant to Ampicillin. Proteus spp is .(١٩٨٩ ,resistant to polymyxin (Cheesbrough :Providencia species .١٫٢٫٦ The genus providencia include three species Providencia aclalifaciens, .(١٩٩٣ P.rettgeri and P. stuartii (Barrow and Feltham DNA hypridization data show that P. alcalifacients can be further divided so that strain that ferment galactose though not adenitol should now be (١٩٨٣ ,.classified as P. prustigianii (Hichman-Brenner et al They are Gram – negative rod, motile, aerobic and facultative anaerobic, catalase positive , oxidase negative,attack sugar fermentatively generally without gas, phenylalanine positive and gelatin not hydrolyze (١٩٩٣,Barow and Feltham) Normal habitat is as same as Proteus sp. The genus grow on selective media, they are non lactose fermenters and some strains give similar reaction to Shigellae on KIA, however they are motile and utilize citrate( .(١٩٨٩,Cheesbrough :Klebsiella species .١٫٢٫٧ Klebsiella pneumoniae was classified into four sub species base on and ١٩٩٤ ,.Holt et al ;١٩٩٣ ,DNA composition (Barow and Feltham .The authors suggested these four subtypes include K .(٢٠٠٠, Cheesbrough pneumoniae.aero, K. Pneumoniae pneumoniae, K. pneumoniae ozoenae and K. pneumoniae rhinosclorimatis. Klebsiella species are thick bacilli often mm in width and it ١٫٠-٠٫٥ mm length and ٣-١ slightly oval vary in size from .(١٩٧٧ ,is non spore forming (Buxton and Frazer Kelbsiella species was widely distributed in nature and gastrointestinal tract of man and animal. Kelbsiella oxytoca has been isolated from faeces of .(١٩٩٧ ,.healthy person (Koneman et al :Kluvera species ١٫٢٫٨ .with two species Kl (١٩٥٦) .This genus was proposed by Asai et al ctrophida and Kl. nocitrophula; presence of polar flagella differentiate it from Escherichia. with two (١٩٨١) .The generic name Kluvera was named by Farmer et al species K. ascorbata and K. cryocrescens. Most of the reported strains have been isolated from sputum and small number from faeces and blood cultures, diarrhoea possibly caused by Kluvera species has been reported (Farmer et al., .(١٩٨١ :Cedecea species .١٫٢٫٩ They are Gram negative, motile, aerobic and facultative anaerobic, catalase-positive and oxidase-negative. Attack sugar fermetitively. Arabinose There are five species described by .(١٩٩٣ ,negative (Barrow and Feltham .(١٩٨١) .Grimond et al :Tatumela species ١٫٢٫١٠ There is only one species, Tatamela ptyseos isolated mostly from .(١٩٨١ ,.sputum (Hollis et al It has small number of flagella , it give inhibition zone around penicillin disk, grow on MacConkey's, most are motile and utilize citrate at .(١٩٩٣ ,C give negative result (Barrow and Feltham°٣٧-٣٥ C, but on°٢٥ :Enterobacter species .١٫٢٫١١ Enterobacter species have many features in common with Klebsiella spp but they are motile but nonmotile are found also, are much the most important clinically. Enterobacter strains produce slightly mucoid colonies, they are found in soil, water and occasionally found in fimbriae ٣ and type ١ human faeces and respiratory tract .They express type also most strain express an aerobactin mediated iron uptake system . Some strain may produce a haemolysin. An outer membrane protein termed Ompx may be pathogenic factor for srain of E. cloacae, this protein appears to reduce production of porins, leading to decreased sensitivity to beta lactam antibiotics and might play a role in host cell invasion (Greenwood et al., .(٢٠٠٢ Enterobacter organisms are Gram – negative motile bacteria, they can be found in the intestinal tract of man and animal, soil, sewage, water and .(٢٠٠٠ ,diary product (Cheesbrough :Morganella species. ١٫٢٫١٢ The genus contain species Morganella morganii, two separate biavar has been identified according to ornithine decarboxlation , one positive and .(١٩٨٩ ,another is negative but some strains are non – motile (Cheesbrough and for Proteus spp is ٪٥٠ The GC content of DNA for M. morganii is .(a, b ١٩٨٤ ,Penner) ٪٤٠ This organism is a quite unimportant but reported that it cause summer .(١٩٩٣ ,diarrhoea in infant (Barrow and Feltham :Edwardsiella species ١٫٢٫١٣ Edwardsiella tarda grow well on ordinary media but produce small C. It is°٣٧ hours incubation at ٢٤ mm in diameter after ١-٠٫٥ colonies of frequently isolated from healthy cold – blooded animals and their environment, human infected from contact these animals, rarely found in faeces of healthy people, but higher isolation rate has been found in patient with diarrhoea, some strains produce a heat – stable toxin (Greenwood et al., .(٢٠٠٢

E. tarda produce H٢S and ferment fewer sugars more slowly and most .(١٩٧٣ ,strain decarboxylase lysine and ornithine (Brenner Edwardsiella, are Gram – negative rods, motile, aerobic and facultative anaerobic, catalase positive, oxidase negative and attack glucose fermentitivly .(١٩٩٣, Barrow and Feltham) :Citrobacter species .١٫٢٫١٤ strains of the genus is reoprted, and regarded them as ٨٠٠٠ In review of a conditional or opportunist intestinal pathogen when conditions were suitable (١٩٧١ ,.for their growth in unusual situation ( Sedlak et al Citrobacter belonged to coliform bacteria but non or late lactose fetrmenetrs, they share certain somatic antigens with Salmonellae, these organism now know as Citrobacter freudii, other species included in the genus, they grow on ordinary media and unpigment mucoid forms are reported (١٩٩٣) .Bettelheim et al .(٢٠٠٢ ,.sometimes occur (Greenwood et al .antigen ٥٧ that C. freundii strain carriers the E. coli O I Citrobacter species are often found in human faeces and may be isolated from variety of clinical specimens and pathogenic mechanisms poorly understood. Strains of C. koseri express type fimbriae and occasional strains .C .(٢٠٠٢ ,.produce form of E. coli verocytotoxin type (Greenwood et al .(١٩٨٩ ,treudnii are late or non lactose fermenter (Cheesbrough Human isolates of all genomospecies except C. koseri have been (١٩٨٥) .Farmer et al .(١٩٩٣ ,.obtained predominantly from stool (Brenner et al reported that C. freundii cause diarrhoea in human. :Hafnia species.١٫٢٫١٫١٥ Hafnia alvei was formerly placed in the genus Enterobacter. Strains of Hafnia spp grow well on general laboratory media, they ferments much narrower range of sugars. Strains of Hafnia alvei isolated from the faeces of man and other animals and are also found in sewage, soil and diary product. They are occasionally opportunistic organism. Some strains carry genes encoding the ability to cause attaching and effacing lesions of intestinal cells .(٢٠٠٢ ,.Greenwood et al) There is a recent evidence to suggest that H. alvei may be emerging ,.Ratnam et al ;١٩٩١ ,Ratnam ;١٩٩١ ,.cause of acute gastroentitis (Albert et al .(١٩٩٢ ,and Wesblom and Millgam ١٩٩٤ ,.Ridell et al ;١٩٧٩ Some strains posses the virulence – associated genes eae A that is responsible for the adherence of organism to epithelial cells and formation of ١٩٩٢ ,.attaching effacing lesions in the intestinal brush border (Albert et al .(١٩٩٤ ,and Law Hafnia species are Gram – negative rod, motile, aerobic and facultative anaerobic, catalase positive, oxidase negative, attack sugar fermentatively, gas is produced, gelatin is not liquefied and urea is not hydrolyzed (Barrow and .(١٩٩٣ ,Feltham :Serratia species .١٫٢٫١٦ Serratia marcessens is the most commonly encountered in clinical specimens and several other specimens. Capsules are not normally formed, most serratia are motile and some strains of S. marcescens produce pigmented colonies on agar, pigmented colonies formed only in presence of oxygen and suitable temperature but in lower temperature growth is poorer and pigment formation is abundant. S. marcescens is widely distributed in nature, but faeces carriage is un common. Serratia spp may express fimbrial haemagglutinin and some strains express cell surface component causing this organism to be high hydrophilic and this may involve in adhesions to eukaryotic cell surface. Serratia spp also express an enterobactin which mediated high affinity to iron uptake system, also toxin resembling E. coli .(٢٠٠٢ ,.verolytotoxin and heat –labile toxin were reported (Greenwood et al Serratia is unique among the Enterobacteriaceae in producing three .(١٩٩٧ ,.hydrolytic enzyme: lipase, gelatinase and DNase (Koneman et al Serratia species are Gram – negative motile rod, they are found mostly .(١٩٨٩ ,in soil and water but rarelly in human faeces (Cheesbrough :Salmonella ١٫٢٫١٧ :Description .١٫٢٫١٧٫١ The salmonellae are short rod usually motile by peritrichous flagella .(١٩٧٤,except S. glinarum and S. pullorum(Buchanan and Gibbons They are Gram – negative, non- sporing, non – capsulated, non

µm in diameter. Mostly ١,٥ ٢,٠µm long and-١,٠ acid fast and measure about occur in singly and occasionally in pair and lent to strain heavier at the poles

,.Salmonella is an intracellular pathogen (Tierney et al .(١٩٧٢ ,Pomeroy) .(٢٠٠٤ ,serotypes (Kauflaman ٢٠٠٠ The genus consist of approximately of The Salmonellae are oxidase, V P and KCN.(٢٠٠٤ .,and Tierney et al ١٩٦٦

negative, catalase and MR positive, produce gas from glucose,produce H٢S from TSI , reduce nitrate and utilize Simmon’s citrate and usually liquefy .(١٩٧٤,gelatin (Buchanan and Gibbons :Classification and nomenclature ١٫٢٫١٧٫٢ Enterobacteriaceae is subdivided into five primary groups of which which contain Salmonella. The genus of ١ Eschericheae represent group species or ٢٠٠٠ Salmonella subdivided into four subgenera which consist of more, the names and antigenic structures of species belonging to subgenera I, .(١٩٧٤ ,II and IV are given in original Kauffman – White Scheme (Buchanan Several system of classification and nomenclature system for Salmonella have been suggested. The genus Salmonella is subdivided into three species S. cholerae. suis as a type of species (Buchanan and (١٩٦٦) Similarly Ewing and Ball .(١٩٤٤ ,.Borman et al ;١٩٧٤,Gibbons suggested same proposal but using S. enteritidis as a type of species. reported that Salmonella is the most (١٩٩٧) .Koneman et al serotype describe in ٢٢٠٠ complex of all enterobacteriaceae with more than Kauffaman – White scheme. In this scheme the Salmonella was grouped A, B, C and so on according to the somatic (O) antigen and subdivided into serotype .and so on according to flagella (H) antigen ٣ ,٢ ,١ different antigenic type of Salmonella. There ٢٠٠٠ There are well over were originally classified as separated species, but now all serotypes are put in single species Salmonella entirica and various subspecies are recognized, but most of serotype infect mammals are found in a subspecies also designated enterica.The full correct designation is for example S. enterica subspecies .(٢٠٠٤ ,.enterica serotype enteritidis (Tierney et al :Pathogenicity ١٫٢٫١٧٫٣ For infection of man large number inoculum of these bacteria are viable (١٠٩-١٠٦) required for induction of human illness, in general between organism is required for infection. Organism ingested in water and other drinks and may be carried through the stomach relatively rapidly, and avoid the effect of gastric acid. Administration of antacid, age less than one year or effect of gastric secretion reduce the infective dose. Bacteria within particle of .(٢٠٠٢ ,.food would also avoid the action of stomach acid (Greenwood et al Once Salmonella enter the lumen of intestine they able to tolerate the action of digestive bile and adhere to gut mucosa and multiply. Certain serotypes such as S. typhimurium express type I fimbirrae which able them to adhere to α mannose - containing molecules on the microvilli of the ileal mucosa, strains of S. enteritiditis are thought to express at least three different Greenwood) (١٤ SEF) ١٤ fimbrial structures including S. entritiditis Fimbriae . (٢٠٠٢ ,.et al Serotype such as S. typhimurium and S. enteriditis also express an adhesion mechanism that does not involve fimbriae. Certain strain of enteric pathogen carry DNA sequences that encode several mechanisms termed pathogenicity islands. S. typhimurium and S. enteritiditis have pathogenicity island that encode an adhesion mechanism comprising both a bacterial adhesin and adhesin receptor which translocated in the host intestine. This process enables these bacteria to insert their own binding site into the gut(Greenwood .(٢٠٠٢ ,.et al Attachment to the host mucosa followed by degeneration of microvilli to form breaches in the cell membrane through which the Salmonella enter the intestinal epithelial cells. Further multiplication in these cells and in macrophage of Payer’s patches follows by certain strains (Greenwood et al., .(٢٠٠٢ Some bacteria penetrate into submucosa and passes to mesenteric lymph nodes. All clinical manifestigation including diarrhoea begin after ideal penetration. Strains of S. typhi infection involves the invasion of blood stream .(٢٠٠٢ ,.and various organs (Greenwood et al Medicinal plants ١٫٣ :(Anise (Pimpinella anisum ١ ١٫٣ Genus: Pimpinella. Species: Pimpinella anisum. Synomous name: Anisum vulgare Gaertn, Anisum officinarum Moench, pimpinola, chylote English name: Aniseed, anise. Arabic name:Yansoon

Anise (P. anisum) is herbaceous, annual herb native to the Mediterranean regions it is cultivated in many places. It is widely cultivated in Europe, Egypt Anise(Pimpinella anisum) is cultivated .(٢٠٠١ ,.and America (Elgayyar et al in Europe, Asia India, Mexico, North Africa, and the USSR. The plant reaches meters and requires a warm and long frost-free growing ٠٫٥ a height of about ٨ The reported life zone for anise production is .(١٫١ days (Fig ١٢٠ season of ٧٫٣ to ٦٫٣ meters of precipitation and a soil pH of ١٫٧ to ٠٫٤ C with˚ ٢٣ to .(١٩٨٤,.Simon et al)

of volatile oil. The pimpinella oil is said to ٪٣-٢ Anise fruits yield the have slightly superior flavor, but most anise oil used is that obtained from of volatile oil, this contain ٪٥-٢٫٥ star – anise. The genuine fruits yield about

-of anethole (C٢H١٢O),chavical, methyl crystal,beta ٪٩٠-٨٠ methoxyphenylacetone (Anisketone), safrole and other minor compounds. The oil is labile to atmospheric oxidation and both anisic aldyhyde and anisic The major .(١٩٥٢ ,and Guenther ١٩٨٩ ,acid are normally present (Evans constituent in oil of anise is anethole. Methylchavicol and para- methoxyphenylacetone are also present, but in lesser relative amounts (Simon Anise and the essential oil extracted from the seeds and foliage . (١٩٨٤,.et al Oil of anise is used as.(٢٠٠٢ ,one are widely used in the food industry (Kolak Oil of.(١٩٥٢ ,and Guenther ١٩٨٩ ,flavorings agent and carminative(Evans anise is used in beverages, confectionaries, pharmaceuticals tooth pastes and mouth wash and medically occasionally employed to stimulate persistalsis .(١٩٥٢ ,Guenther) Anise is used as a carminative, antiseptic, antispasmodic, expectorant, stimulant, and stomachic. In addition, it has been used to promote lactation in nursing mothers and as a medicine against bronchitis, indigestion and lice. Oil of anise is used today as an ingredient in cough medicine and lozenges and is reported to have diuretic and diaphoretic properties. If ingested in sufficient quantities, anise oil may induce nausea, vomiting, seizures, and pulmonary edema. Contact of the concentrated oil with the skin can cause .(١٩٨٤,.irritation(Simon et al The essential oils extract from seeds of seven spices including Pimpinella anisum have been studied for antimicrobial activities against eight pathogenic bacteria including , Pseudomonas aeruginos, E.coli and Klebsiella spp. These oils are equally effective when compared .(٢٠٠١ ,.with standard antibiotics at very low concentrations (Singh et al plants were prepared and tested against ٣٣ The methanolic extraction of bacteria including Escherichia coli, Pseudomonas aeruginosa, Serratia ١١ crude ١٨ marcescens and others to evaluate antimicrobial properties of from plant species including P. anisum. It was found to possess (٪٢٧) extracts antimicrobial activity against one or more tested microorganism, and the study showed that water soluble part of methanolic extracts were found to be more effective than the water insoluble ones against the microorganism tested .(٢٠٠٠ ,.Sokmen et al) The in vitro antimicrobial activity of hyrosols (distilled spice water) of sixteen species including anise were tested on fifteen bacteria including E. coli ,٢٥٩٢٢ Escherichia coli ATCC ,٢٥٣١ Enterobacter aerogenes CCM ATCC Salmonella eneritidis, S. gallinarum, S.typhimurium and H٧ :٠١٥٧ the hyrosols of five spice including anise ,١٥٠١ Yersinia enterocolitica ATCC had antimicrobial activities against some of the tested organisms, but not all of .(٢٠٠٣ ,the tested bacteria (Sagdic and Ozcan plant including Pimpinella anisum ١٢ The antimicrobial activity of were examined against some pathogenic bacterial (Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Shigella flexineri, Salmonella typhi, Enterobacter aerogenus and Staphylococcus aureus) these plant give good .(٢٠٠١ ,.result against these microorganisms (Larhsini et al Essential oil (volatile oil) of anise was collected and tested against some bacteria including Escherichia coli, Pseudomonas aeroginasa, and some mm to bacteria but highly inhibitory to ٢٥ fungi, it gave inhibition zone about Antibacreial activity of essential .(٢٠٠١ ,moulds more than bacteria (Elgayyar oil (volatile oil) of anise was investigated against L.monocytogen and S.enteritidis. The result revealed that S.enteritidis was particulary sensitive to ihibition by combination anise essential oil with methyl-parben(Fyle et al.,). The constituents of many volatile oils are stated to interfere with .(١٩٨٩,respiration and electron transport in a variety of bacteria (Evans :Hydnora abissynica ١٫٣٫٢ Family: Hydnoranceae. Genus: Hydnora. Species: Hydnora abissynica. Synomous name: Hydnora africana. A rabic name: Tartous. This plant grows as parasites on the root of Acacia spp. The plant is distributed in central Sudan. It΄s chemical constituent are not well known. In folkloric medicine, the whole plant is used for swelling tonsillitis and .(١٩٩٧ ,.dysentery (Kamal et al The under ground stem which like a root is normally bioled used as cure for throat, dysentry and removing of the placenta if not come out on time .(١٩٧٦ ,Kokwaro) ,repoted that the plant named Hydnora africana (١٩٦٢) Watt and Preyer is a parasite growing principally on the root of Eurobia mauritania and on Euphorbia capwimedusae at the cap and on the acacia trees. Significant antibacterial activity were obtained from Hydnora abissynica when examined against Escherichia coli, Pseudomonas .(٢٠٠١ ,.aeruginosa and Staphylococcus aureus (Elegami et al

Anise whole plant :١٫١ Figure

(Anise (Pimpinella anisum)fruit (seeds :١٫٢ Figure

Hydnora abissynica :٢ Figure CHAPTER TWO MATERIALS AND METHODS .٢

:Asepsis and sterilization ٢٫١ :Flaming ٢٫١٫١ It was used to sterilize slides, coverslips and glassrods. :Red heat ٢٫١٫٢ It was used to sterilize loop wire, needles and points searing spatulas by holding them over Beunsen burner flame until became red hot. :Hotair oven ٢٫١٫٣ It was used to sterilize metals, glass wares such as test tubes, graduate pipettes, flask, Petri dishes and cotton swabs. The holding period was one hour .C°١٨٠ and temperature was :C°١٠٠ Steaming at ٢٫١٫٤ Repeat steaming (tyndalization) was used for sterilization of sugar and media that could not be autoclaved without determintal effect to their .(١٩٧٥) .constituents. It was carried out as described by Cruickshank et al :(Moist heat (autoclave ٢٫١٫٥ Media, solutions and plastic wares were sterilized by autoclaving at .minutes ١٥ for (١٥Ib/inch٢)C°١٢١ :Irradiation ٢٫١٫٦ minutes was used to sterilize media ٢٠ Ultraviolet irradiation for preparation room. :Disinfection ٢٫١٫٧ alcohol were used for disinfecting the floor ٪٧٠ Phenol disinfectant and and working preparation room in the laboratory. :Reagents and indicators ٢٫٢ :Reagents ٢٫٢٫١ Most reagents were obtained from British Drug House (BDH). :Sodium chloride /normal saline ٢٫٢٫١٫١ Normal, physiological, or isotonic saline was prepared as described in grams of sodium chloride into one litre of ٨٫٥ Oxiod manual by dissolving concentration ٪٠٫٨٥ distilled water to obtain

:(Hydrogen peroxide (H٢O٢ ٢٫٢٫١٫٢ This was obtained from British Drug House (BDH). It was prepared as .aqueous solution and it was used for catalase test ٪٣ :Potassium hydroxide ٢٫٢٫١٫٣ ٪٤٠ This was obtained from Hopkin and Willians, it was prepared as .and it was used for V P test (١٩٩٣) solution according to Barrow and Feltham :Kovac’s reagent ٢٫٢٫١٫٤ ,٥g of para - dimethyl amino benzaldyhyde This reagent composed of .ml concentrated hydrochloric acid ٢٥ ml amyl alcohol and ٧٥ :Lead acetate ٢٫٢٫١٫٥ long were impregnated in ٦٠-٥٠ mm wide and ٥–٤ ,Filter paper strips lead acetate saturated solution and then dried and it was used for hydrogen sulphide test. :Potassium hydroxide and hydrochloric acid ٢٫٢٫١٫٦ Potassium hydroxide and hydrochloric acid were used for adjusting pH. :Methyl red solution ٢٫٢٫١٫٧ Methyl red manufactured by British Drug House (BDH) was used. The by (١٩٩٣) solution was prepared as described by Barrow and Feltham ml ethanol and volume was made up ٤٠ gram of methyl red in ٠٫٠٤ dissolving .ml with distilled water ١٠٠ to :α – naphthol solution ٢٫٢٫١٫٨ α – naphthol was obtained from Hopkin and Willian, London and the solution as descried by Barrow and Feltham ٪٥ solution was prepared as .The solution was used for V P test .(١٩٩٣) :Tetramethyl – p – phenyline diamine diahyrochloride ٢٫٢٫١٫٩ This reagent was obtained from Hopkin and Williams, London. It was .aqueous solution and it was used for ٪١ prepared as :Indicators ٢٫٢٫٢ :Bromothymol blue ٢٫٢٫٢٫١ This was obtained from BDH, the solution was prepared by dissolving .ml distilled water ١٠٠ g of bromothylmol blue powder in ٠٫٢ :Andrade’s indicator ٢٫٢٫٢٫٢ g, distilled water one litre and N – NaOH ٥ It composed of acid fuchsin by (١٩٩٣) ml. It was prepared as described by Barrow and Feltham ١٥٠ ml of alkali solution is ١٥٠ dissolving the acid fuchsin in distilled water then hour with ٢٤ added, mixed and allowed to stand at room temperature for frequently shaking until the colour changed from red to brown. :Bromocrysol purple ٢٫٢٫٢٫٣ ٦٣ N- NaOH to ٠٫٠٥ ml of ٣٧ was added to (٪٠٫٢) Bromocrysol purple ml distilled water. :Gram stain reagents ٢٫٢٫٢٫٤ :Lugol’s iodine ٢٫٢٫٢٫٤٫١ Formula: g ٢٠ Potassium iodide g ١٠ Iodine litre ١ Distilled water

Potassium iodide was weighed and dissolved in about quarter of water. Iodine was added to potassium iodide solution and mixed well. The solution was made up to one litre with distilled water, mixed well and then stored in dark place at room temperature.

:Crystal violet ٢٫٢٫٢٫٤٫٢ g ٢٠ Crystal violate g ٩٫٠ Ammonium oxalate ml ٩٥ Ethanol Up to

Crystal violet was weighed and alcohol was added, mixed well until the dye was completely dissolved. Ammonium oxalate was weighed and ml of distilled water, added to stain and made to one ٢٠٠ dissolved in about litre with distilled water and mixed well and then stored at room temperature. :Acetone – alcohol decolorizer ٢٫٢٫٢٫٤٫٣ Formula: ml ٥٠٠ Acetone ml ٤٧٥ Ethanol or methanol, absolute ml ٢٥ Distilled water

Distilled water was mixed well with alcohol, acetone was measured and was added immediately to alcohol solution, mixed well and stored at room temperature. : Diluted carbol fuchsin ٢٫٢٫٢٫٤٫٤ volumes of ٢٠-١٠ One volume of strong carbol fuchsin was added to distilled water to prepare diluted carbol fuchsin . Strong carbol fuchsin consists of two solutions: ml of ethanol ١٠٠ Solution A: Ten grams of basic fuchsin mixed with -١ .C overnight° ٣٧ and dissolved in stoppered bottle and kept at(٪٩٥) ١٠٠ml of distilled water and Solution B: Five grams of phenol mixed with-٢ dissolved. ml of solution A ١٠ Strong carbol fuchsin was prepared by pouring .ml of solution B ١٠٠ poured into

:Equipments used ٢٫٣ a. Thermostatically controlled water bath. b. Thermostatically controlled incubator. c. Microscope. e. Glass ware: Petri dishes, disposable Petri dishes, test tubes, Pasteur pipettes, graduated pipettes (different sizes), graduated slenders, different size flasks, bottle (Bijou, universal) and slides. f. Hotair oven. g. Digital pH meter to adjust pH. h. Steamer. i. Autoclave. :Antibiotic used ٢٫٤ In this study six antibiotics were chosen which include Gentamycin ,(٥٠µl/٢٥µg) ٥٠µl), Sreptomycin/٣٠µg) ٥٠µl), Nalidixic acid/١٠µg) .(٥٠µl/٢IU) ٥٠µl) and Penicillin/٢٥µg) ٥٠µl ), Tetracycline/٢٥µg) Ampicillin Nutrient broth was used as diluents to reach standard concentration. :Culture media ٢٫٥ :Liquid media ٢٫٥٫١ :(MR test medium (Glucose – phosphate medium ٢٫٤٫١٫١ ٥g Peptone

٥g K٢HPO٤

٥g Glucose

The solid substances were dissolved in one litre of distilled water, the ١٫٥ml volumes in test tube and then distributed into ٧٫٢ pH was adjusted to .minutes ١٠ for (١٠Ib/inch٢) C°١١٠ and sterilized by autoclaving at

: V P test medium ٢٫٥٫١٫٢

g ١٠ Peptone g ٥ Glucose ml ١٠٠٠ Distilled water

The medium ingredients were mixed and dissolved by gentle heating, and distributed in test tubes and sterilized by ٧٫٤ the pH was adjusted to .minutes ١٠ for (Ib/inch٢ ١٠) C°١١٠ autoclaving at :Peptone water ٢٫٥٫١٫٣ .(١٩٩٣) This medium was prepared as described by Barrow and Feltham grams of sodium chloride were dissolved into one ٥ Ten grams peptone and The medium was .٧٫٢ litre of distilled water and the pH was adjusted to distributed into five ml amount in universal bottles and test tubes and was then .(١٥Ib/inch٢) C°١٢١ minutes at ١٥ sterilized by autoclaving for :Nitrate medium ٢٫٥٫١٫٤ g) were dissolved into ٥٫٠) g) and peptone ٠٫٢) Potassium nitrate ml amount ٥ ml distilled water . The medium mixture was distributed in ١٠٠٠ .(١٥Ib/inch٢) C°١٢١ minutes at ١٥ in test tubes and then autoclaved for :Nutrient broth ٢٫٥٫١٫٥ This medium was obtained from Oxoid(Ltd). The prepared medium ٢) g) and yeast extract ٥) g), lamb lemco powder ٥)contain sodium chloride g). It was prepared according to manufacturer’s instructions. Thirteen grams of powder was dissolved in one litre of distilled water and distributed ml amounts in flasks ٢٠٠ ml and ١٠٠ into five ml volumes in test tubes and .minutes ١٥ for(١٥Ib/inch٢) C°١٢١ and sterilized by autoclaving at :Peptone water sugar ٢٫٥٫١٫٦ Peptone water sugar medium was prepared as described by Barrow and ml, Andrade’s indicator ٩٠٠ It contained peptone water .(١٩٩٣) Feltham ml. The pH of peptone ٩٠ ml and distilled water ١٠ ml, sugar solution ١٠ .before the addition of Andrade’s indicator ٧٫٣-٧٫١ water was adjusted to ml into ٢ The complete medium was mixed then distributed into portion of clean test tubes containing Durham’s tube and sterilized by autoclaving at ,minutes .Sugar tested were sucrose, adenitol, arabinose ١٠ Cfor°١١٥ inositol lactose, maltose, raffinose rhamm-nose, salicin sorbitol, xylose, mannitol :Selenite- F- broth ٢٫٥٫١٧ This medium contained sodium hydrogen selenite (sodium selenite) ,g), di – sodium hydrogen phosphate ٠٫٨) g), mannitol ١٫٠) g), peptone ٠٫٨)

(ml ٢٠٠) g) and distilled water ٢٫٠) anhydrous Na٢HPO٤ This medium was prepared with care, the dry ingredients were mixed in C to dissolve the solids, the pH was adjusted to°٨٠-٧٥ water and heated to ml amounts in screw – cap universal bottles, after ٥ and then dispensed in ,٧٫٠ minutes. The sterilized medium was ٢٠ that was sterilized by steaming for stored in cool dark place. :Semi solid media ٢٫٥٫٢ :Oxidation – Fermentation (O/ F) medium ٢٫٥٫٢٫١ g), sodium chloride ٢٫٠) This medium contained tryptone or peptone

g), agar ٠٫٣) g), di – potassium hydrogen phosphate, anhydrous K٢HPO٤ ٥٫٠) .litre ١ ml) and distilled water ٣) (w/v ٪١) g), bromothymol blue solution ٢٫٥) by (١٩٩٣) The medium was prepared according to Barrow and Feltham ,٧٫١ dissolving the solids in one litre of distilled water and pH was adjusted to ml ١٠ then the indicator was added The medium was then distributed into .minutes ١٠ C for°١١٥ volumes into test tubes. sterilized by autoclaving at :Carry – Blair transport medium ٢٫٥٫٢٫٢ g), di- sodium ٠٫٧٥) This medium contained sodium thiogycolate

٢٫٥) g), agar ٢٫٥) g), sodium chloride ٠٫٥٥) (hydrogen phosphate (Na٢HPO٤ ml ٤٩٥ ٤٫٥ml) and distilled water) (w/v ٪١) g), calcium chloride solution by dissolving the (١٩٨٩) It was prepared as described by Cheesbrough dry ingredients in water by heating, then the medium was allowed to cool to ml of freshly prepared calcium chloride solution were ٤٫٥ C and then°٥٠ (١٠/N) ١/mol ٠٫١ using ٨٫٤ added and mixed well. The pH was adjusted to ml volumes in screw – cap ٧ sodium hydroxide, the medium was dispensed in ١٥ ml capacity, then sterilized by steaming (with cap loosen) for ١٠ bottle of minutes, once cooled the caps were tightened, then labeled and stored in dark cool place. :Motility medium – Gragie tube medium ٢٫٥٫٢٫٣ g and agar (Oxoid ١٣ (This medium contained nutrient broth (Oxoid .g ٥ (١ .agar No .(١٩٩٣) The medium was prepared as described by Barrow and Feltham g of agar and ٥ Thirteen grams of dehydrated nutrient broth were added to this ,٧٫٤ were dissolved in one litre of distilled water. The pH was adjusted to ml test tubes containing the ٢٠ ml into ٥ medium was dispended in volume of appropriate Gragie tube then the medium in test tubes were sterilized by .minutes ١٥ C for°١٢١ autoclaving at :Solid media ٢٫٥٫٣ :MacConkey's agar ٢٫٥٫٣٫١ g), lactose ٢٠) This medium contained peptic digest of animal tissue g), neutral ٠٫٠٠١) g), crystal violet ٥) g), sodium chloride ١٫٥) g), bile salt ١٠) .(g ١٥) g) and agar ٠٫٠٥) red Fifty two grams of dehydrated powder were added to one litre of distilled water, mixed and boiled in the water bath to dissolve the ingredients minutes and poured ١٥ C for°١٢١ completely, then sterilized by autoclaving at ml amount, the poured plates were left to solidify ١٥ into sterile Petri dishes in at room temperature on flat surface. :Diagnostic sensitivity test (DST) agar ٢٫٥٫٣٫٢ This medium was purchased from Oxoid(Ltd).The medium ,(g ٢٫٠) ١٠٫٠g), veal infusion) (٤٦ contianed protease peptone (Oxoid L g), adenine sulphate ٢٫٠) g), di – sodium phosphate ٣٫٠) sodium chloride ,(٠٫٠١g) g), uracil ٠٫٠١) ٠٫٠١g), xyanthine) ٠٫٠١g),gauanine hydrochloride) .(g ١٢٫٠) ٢ .g) and agar No ٠٫٠٠٠٠٢)anuarine The medium was prepared according to manufacture’s instructions by g in one litre of distilled water, then brought to boil to dissolve ٤٠ dissolving ,minutes ١٥ C for°١٢١ completely after that it was sterilized by autoclaving at ml amount and allowed to solidify on ١٩ then poured into Petri dishes in leveled surface. :(Kligller's iron agar (KIA ٢٫٥٫٣٫٣ ,(g ٢٠٫٠) This medium composed of casein enzyme hydrolyzate g), lactose ٥٫٠) g), sodium chloride ٣٫٠) g), yeast extract ٣٫٠)beef extract g), ferrous ٠٫٣) g) , sodium thiosulphate ١٫٠) (g), glucose (anhydrous ١٠٫٠) .(g ١٥٫٠) g) and agar ٠٫٢٥)g), phenol red ٠٫٢) sulphate g) was suspended in one litre of distilled ٥٧٫٣) Dehydrated medium water, then boiled to dissolve the medium completely. The dissolved medium ml amount, then sterilized by autoclaving ١٠ was dispended into test tubes in minutes. These tubes were allowed to cool in ١٥ for (Ib/inch٢ ١٥) C°١٢١ at inclined position to form slope with about one inch butt. :Nutrient agar ٢٫٥٫٣٫٤ This medium was purchased from Oxoid (Ltd). It contained lamb – ,(g ٢٫٠) (٢٠ g), yeast extract (Oxoid L ١٫٠) (٢٩ lemco powder (Oxoid L Oxoid) ٣ .g) and agar No ٥٫٠) g ), sodium chloride ٥٫٠) (٣٧ peptone (Oxoid L .(g ١٥٫٠) (١٣ L Twenty eight grams of dehydrated medium were dissolved in one litre of distilled water, then bruoght to the boil to dissolve completely, after that the minutes, then cooled to ١٥ C for°١٢١ medium was sterilized by autoclaving at ml amount per sterile Petri dish, the poured ١٥ Cand distributed in°٥٠ about plates were left to solidify at room temperature on the leveled surfaces. Also ٥ml amount Bijou bottle and allowed to the medium was distributed in solidify in slope position to form slope and butt. :Nutrient gelatin ٢٫٥٫٣٫٥ Nutrient gelatin medium was purchased from Oxoid (Ltd). It .(g ١٢٠٫٠) g) and gelatin ٥٫٠) g), peptone ٣٫٠) contained beef extract The gelatin was added to one liter of distilled water and allowed to minutes to dissolve gelatin. The other ingredients were added ٣٠-١٥ stand for and then heated to boil to dissolve other constituents and the pH was adjusted The medium was distributed into screw - cap bottles and sterilized by .٧٫٠ to .minutes ١٥ C for°١٢١ autoclaving at :(١٠٨ Salmonella – Shigella (SS) agar (M ٢٫٥٫٣٫٦ The medium was obtained from Himedia Company in dehydrated form. g), bile ٥٫٠) g), beef extract ٥٫٠)It composed of peptic digest of animal tissue g), neutral red ١٠٫٠)g), sodium citrate ١٠٫٠) g), lactose ٨٫٥)salt mixture .(g ١٥٫٠) g) and agar ٠٫٠٠٠٣٣) g), brilliant green ٠٫٠٠٢٥) The medium was prepared according manufacture instructions by grams in one litre of distilled water, and then heated to boil ٦٣٫٠ dissolving with frequent agitation to dissolve the medium completely, cooled to about C, mixed well and poured into Petri dishes and lefted to solidify at room°٥٠ temperature on leveled surface. :Simmon’s citrate agar ٢٫٥٫٣٫٧ The dehydrated medium was obtained from Difco (Ltd). It composed

g), sodium citrate (tri ٠٫٨) g), NaNH٤PO٤ ٠٫٢) g), NH٤H٢PO٤ ٢٫٠) of MgSO٤ .(g ٠٫٠٨) g) and bromothymol blue ١٥)٣ .g) agar No ٠٫٠٨) (basic Twenty – three grams of the dehydrated medium were suspended in one litre of distilled water, brought to boil to dissolve completely, the pH was .minutes ١٥ C for°١٢١ and was sterilized by autoclaving at ٧٫٠ adjusted to ml each and ٥ The medium was then dispended into Bijou bottles in portion of left to solidify in slope position. :Urea agar base ٢٫٥٫٣٫٨ Urea agar base was bought from Oxoid (Ltd). It composed of peptone (١٣ Oxoid L) ٣ .g), phenol red agar No ١٫٠) g), dextrose sodium chloride ١٫٠) g) and potassium dihydrogen phosphate ١٫٢)g),di-sodium phosphate ٠٫٠١٢) .(g ٠٫٨) The medium was prepared according to manufacturer instructions by ml of distilled water, and dissolved by boiling, then ٩٥ gram in ٢٫٤ dissolving ٥ C then°٥٠ minutes and cooled to ١٥ C for°١٢١ sterilized by autoclaving at were added under aseptic (٢٠ urea solution (Oxoid SR ٪٤٠ ml of sterilized condition. The complete sterilized medium was distributed into sterile Bijou .ml amount and allowed to set in slope position ٥ bottle in :(Ammnonium salt sugars (ASS ٢٫٥٫٣٫٩ Ammonium salt sugars (ASS) were prepared as described by

,(g ٠٫٢) g) KCl ١٫٠) ٢HPO٤(It contained (NH٤ .(١٩٥٢) .Smith et al

١٠٠٠)٢٠g), distilled water) g), agar ٠٫٢)g), yeast extract ٠٫٢)MgSO٤٫٧H٢O .ml ٠٫٠٤ ( ٪٠٫٢ ) ml) and bromocrysol purple solution Solids were added to water and dissolved by steaming then indicator min. The basal ٢٠ C for°١١٥ was added and sterilized by autoclaving at C and appropriate carbohydrate was°٦٠ medium was allowed to cool to about mixed and ,٪١ added as a sterilized solution to give final concentration distributed aseptically into sterile test tubes which were inclined so that the medium set in slope and butt. :Collection and transport of sample -٢٫٦ Fify-one faecal samples were collected from infants and children stool suffering from severe dirrahoea from Omdorman Pediatric Hospital. For collection of sample sterile plastic container of sufficient size and having a tight fitting leak proof lid were used. The collected stool was transported to the laboratory, it was processed immediately or as soon as possible, but not Full swab .(١٩٨٧) more than two hours after collection as adviced by WHO was taken from stool container and immersed in bottle of Carry – Blair transport medium. Sample from each container of stool was examined immediately for general properties such as consistency, colour, presence of protozoa, helminthes, mucus, pus cells and chemical reaction. Any sample revealed the presence of protozoa or the helminthes was excluded. :Culture of specimens ٢٫٧ The fifty-one collected faecal samples were inoculated onto MacConkey's agar, and into selenite broth medium. The inculcated two media hours. The growth from Selenite -F -broth was ٢٤ C for°٣٧ were incubated at .hours ٢٤ C for°٣٧ subcultured on Salmonella Shigella agar and incubated at After incubation periods the colonies characteristics were observed and smear was made from each type of colony. The smear was dried into air and then fixed by heating, stained by Gram's staining methods as described by Barrow The stained smear was examined under light microscope .(١٩٩٣) and Feltham for cell morphology and staining reaction. All isolated bacteria were purified by several subculturing from single well – separated colony. The purity of the culture was checked by examining Grams stain smears. The pure culture was then used for studying cultural and biochemical characteristic, antibiotics and extracts of medicinal plant sensitivity for the isolated bacteria. :Microscopic examination ٢٫٨ Smear was made from each type of colony on primary culture and from purified colonies, fixed by heating and stained by Gram's method as described Then the stained smear was examined .(١٩٩٣) by Barrow and Feltham microscopically by oil immersion lens. The smear was examined for cell morphology and staining reaction. :Identification of bacteria ٢٫٩ The purified isolates were identified according to criteria described by This included staining reaction, organism .(١٩٩٣)Barrow and Feltham morphology, growth condition, and the colony characteristics on different media, motility and biochemical characteristics. :Biochemical method ٢٫١٠ :Catalase test ٢٫١٠٫١ A .(١٩٩٣) The test was carried out as described by Barrow and Feltham

was placed on clean slide and a colony of tested organism H٢O٢ ٪٣ drop of ٪٣ cultured on nutrient agar was picked by glass rod and added to the drop of

A positive result was indicated by production of air bubbles .H٢O٢ immediately, no bubbles indicated negative result. :Citrate test ٢٫١٠٫٢ The organism grown in nutrient agar was heavily inoculated into slope hours ٢٤ C, examined after°٣٧ of Simmon’s citrate agar, then incubated at days. Blue colour and growth of the ٧ incubation and examied daily for organism indicated positive result; green and no growth indicated negative result. :Urease test ٢٫١٠٫٣ A slope of Christensen’s urea agar medium was incubated heavily with hours and examied daily ٢٤ C, examined after°٣٧ test organism, incubated at days. Red – pink color indicated positive result, no red – pink or yellow ٥ for color indicated negative result. :Oxidase test ٢٫١٠٫٤ .(١٩٩٣) The test was carried out as described by Barrow and Feltham solution of tetra methyl – p – phenylene ٪١ Strip of filter paper was soaked in diamine dihyrochloride (oxidase reagent) and dried in hot air oven and then placed on clean glass slide by forceps. A fresh young tested culture on nutrient agar was picked off with sterile glass rod rubbed on filter paper strip. If blue seconds, the reaction was considered ١٠-٥ purple color developed within positive. No blue – purple color indicated negative result. :Sugar fermentation test ٢٫١٠٫٥ .(١٩٩٣) The test was carried out as described by Barrow and Feltham ,hours peptone water cultures ٢٤ Carbohydrate medium was inoculated with days. Production of acid was ٧ C and examined daily for°٣٧ then incubated at indicated by appearance of reddish or pink color, while gas production was indicated by the presence of empty space in the inverted Durham’s tubes. :Indole test ٢١٠٫٦ Indole production test was carried out as described by Barrow and The test organism was cultured into peptone water which .(١٩٩٣) Feltham hours. One milliliter of ٤٨ C for°٣٧ contain tryptophan and incubated at p-dimethylamine benzaldehyde was run-٤ Kovac’s reagent which contained down along side of the test tube. Appearance of pink color in the reagent layer within a minute indicated positive reaction. :Oxidation – fermentation (O/F) test ٢٫١٠٫٧ .(١٩٩٣)The test was carried out as described by Barrow and Feltham The test organism was inoculated by stabbing with straight wire loop into duplication test tubes of Hugh and Leifsons medium. To one of the test tube layer of melted soft paraffin oil was added to the medium to seal it from air, C and were examined daily for°٣٧ both inoculated test tubes were incubated at two weeks. Yellow color in open tube only indicated oxidation of glucose, yellow color in both tubes showed fermentation reaction and green or blue color in open tube and yellow color in sealed tube indicated production of alkali and green in both tube indicated no oxidation and no fermentation of glucose. :Methyl red (MR) test ٢٫١٠٫٨ Glucose .(١٩٩٣) It was carried out as described by Barrow and Feltham hours ٢٤ phosphate peptone water (MR) medium were inoculated with – hours. Two drops of ٢٤ C for°٣٧ peptone water culture then incubated at methyl red reagent were added, shaken well and examined. Appearance bright red color indicated positive reaction whereas orange yellow color indicated negative reaction. :Gelatin hydrolysis test ٢٫١٠٫٩ Nutrient gelatin in screw – capped bottle was inoculated by stabbing C for two weeks and examined°٣٧ with straight wire loop and incubated at -C in refrigerator. Un°٤ days for liquefaction after cooling at ٣-٢ every inoculated control bottle was included.

:Hydrogen sulphide (H٢S) production test ٢٫١٠٫١٠ This test was performed by two methods: a. Production in Kiligller ion agar (KIA). A tube of KIA was inoculated by stabbing the butt and streaking the slope, days for ٧ C. The tubes were observed daily up to°٣٧ then incubated at

.blacken of the butt which indicated H٢S production

b. H٢S production was also examined by lead acetate paper method, A tube of peptone water medium was inoculated by test organism and lead acetate paper was inserted between the cotton plug and tube, C and examined daily for a week, blackening of the°٣٧ incubated at paper was considered positive result. :Nitrate reduction test ٢٫١٠٫١١ .(١٩٩٣) This test was carried out as described by Barrow and Feltham ٢٤ C for°٣٧ The test culture was inoculated into nitrate broth and incubated at ml of solution A followed by one ml of solution B of the nitrate ١ hour, then test reagent were added. After few minutes development of red color was observed. If red color didn’t develop zinc was added to see whether there was residual nitrate or not. Red color development indicated reaction by zinc but not by organism, whereas unchanged color indicated nitrate in original medium had been reduced completely and nitrite was further broken down by organism. :Voges – Proskaur (VP) test ٢٫١٠٫١٢ .(١٩٩٣) The test was performed as described by Barrow and Feltham The test culture was inoculated into glucose phosphate medium and incubated (potassium hydroxide (KOH ٪٤٠ hours. Three milliliter of ٤٨ C for°٣٧ at ١٥ aqueous solution were added, it was well shaken and examined after minute and one hour, when bright pink color developed, the reaction was regarded as positive indicated production of acetyl – methyl carbinol. :Motility test ٢٫١١ The Gragie tube in semisolid nutrient agar was prepared as A small piece of colony of the .(١٩٧٥) .described by Cruickshank et al bacterium under test was picked by the end of straight loop and inoculated into .C overnight°٣٧ of semi – solid agar in the Gragie tube and then incubated at The organism was considered motile if there was turbidity in and outside of the Gragie tube. . :Staining method ٢٫١٢ was (١٩٩٣) Gram staining method described by Barrow and Faltham used. .Smear was fixed by heating by passing over the flame three times .١ .minute ٣-٢ Crystal violet was added to smear for .٢ .Washed with distilled water .٣ .Lugol’s iodine was added for one minute .٤ .seconds ٣-٢ Decolorized with acetone – alcohol for .٥ .Washed with distilled water .٦ .Counter stained with diluted carbol fuchsin for half minute .٧ .Washed with water .٨ Dried with filter paper and examined under microscope using the oil .٩ immersion objective lens. Gram- positive bacteria appeared purple while Gram- negative bacteria appeared red. :Plants extraction methods ٢٫١٣ :Extraction of Hydnora abissynica ٢٫١٣٫١ :Water extraction ٢٫١٣٫١٫١ Water is almost universal solvent used for extraction of plant. In this study water was used to extract the active ingredient of Hydnora abissynica. ml of ١٠٠ grams of H. abissynica were soaked in ٢٠ a. Amount of hours. The content of the ٢٤ sterile distilled water in sterile flask for .C for later use°٤ flask were then filtered, the filtrate was kept at ml sterile ١٠٠ b. Twenty grams of H. abissynica were soaked in distilled water and placed into sterile flask and put into water bath at hours. The content of flask was then filtered and the ٤-٣ C for°٨٠-٧٠ .C for later use°٤ filtrated was kept at c. The plant was extracted by the same method as b, but after filtration of flask content, the filtrated was dried at room temperature in solutions were prepared from the ٪٢٠ and ٪١٠ sterile Petri dishes then dried material. :Petroleum ether extraction ٢٫١٣٫١٫٢ In this study petroleum ether was used as solvent. Finely ground sample, from the plant was accurately weighed, and put in empty thimble .The thimble was covered with soxhlet extraction apparatus. .Pre-weighed ml of petroleum ether was fitted to the ٤٠٠-٣٥٠ round bottomed flask full of ١٢h extractor, the apparatus was assembled, the extraction continued for .The apparatus was evaporated first at room temperature then dry in an oven ١٠٥ºC. The oil constant weight was expressed as percentage of the oil at content. The non polar extract was calculated as follow: Oil % = The weight of oil extracted (g) x١ Weight of sample :Anise (Pimpinella anisum) volatile oil extrataction .٢٫١٣٫٢ Volatile oils are usually obtained by distillation of plant part containing the oil. The methods of distillation depend on the condition of the .(١٩٩٦ ,.plant material and properties of volatile oil (Robber et al To obtain volatile oil from anise water distillation technique was used as Water and anise seeds were put at the base of the . (١٩٨٩)discribed by Evans .hours ٦ still which was heated by steam coils. The distillation took about The distillate which consists of a mixture of oil and water was condensed and collected in a suitable receiver which was a large glass jar with one of outlet near the base and another near the top. The distillate separated into two layers. The oil was withdrawn through the upper outlet and the water from the lower outlet because the water is heavier than the oil.Then the oil collected in and ٪١٫٠ ,٪١٠) suitable container after that different concentration was made .The diluent used was methanol .(٪٠٫١ :Cup plate method ٢٫١٤ Cup plate Method was performed as described by Cruichshank et al. ٥ml ml) of tested organism was inoculated into ٠٫٠١) Standard loopful .(١٩٧٥) ml of sterile normal ٥ h, then ٢٤ ٣٧ºC for nutrient broth and incubated at saline solution were added to the culture. This saline suspension was ml of this saline suspension were ٢ cells per ml. About ١٠٦-١٠٥ equivalent to transferred with sterile Pasteur pipette onto DST agar plate and the whole plate surface was covered by tipping the plate in different direction. Excess ٣٠ C for°٣٧ fluid was removed using sterile pipette and the plate was dried at minutes in the incubator, then cups were made by cutting with a cork borer and then the cut agar was removed carefully so that the edge of the cup was not lifted. These cups were filled with extracts of plants or antibiotic (by hours. The diameter ٢٤ Cfor°٣٧ ٥٠µl in each cup), then incubated at pouring of growth inhibition zone was measured in millimeter and then scored as (+) mm ٢٠ :(++++) ;mm ١٥:(+++) ;mm ١٠:(++) ;mm ٥ when inhibition zone was and (-) when the inhibitor zone was not noticed.

CHAPTER THREE RESLUT .٣ :Isolation and identification ٣٫١

Fifty-one faecal samples were collected randomly from children below five year of age suffering from diarrhoea in Omdurman Pediatric Hospital. The samples were inoculcated onto appropriate deferential and selective media (MacConkey's’s agar medium, selenite –F- broth medium and Shigella and ٣٧oC. All Salmonella (SS) agar medium) and were incubated aerobically at specimens showed bacterial growth. Eighty nine bacterial isolates were obtained from all specimens. Thirty-eight specimens gave mixed growth whereas thirteen specimen gave one type of growth. These colonies were picked up and purified by subculturing three times on non selective medium (nutrient agar). The bacterial isolates were identified according to their cultural characteristics, microscopic appearance, Gram’s staining reaction and (١٩٩٣,biochemical properties(Barrow and Felam :Main findings ٣٫٢ All bacteria isolates obtained from faecal samples in this study were and ١ identified. Different bacterial species were recovered as shown in table -: The following bacteria were isolated and identified .٣ figure :Escherichia coli isolates ٣٫٢٫١ of Escherichia coli were isolated in this (٪٧٠٫٩) Thirty-six strains .(٣ and Figure ١ study (Table :Cultural characteristics ٣٫٢٫١٫١ On MacConkey's agar medium, large rose coloured colonies were On Salmonella Shigella .(٧ observed indicating lactose fermentative (Figure (SS) agar lactose fermenting colonies were seen. :Microscopic Examination ٢ .٣٫٢٫١ Gram-negative rods, non-spore forming cells were observed :Biochemical reaction ٣٫٢٫١٫٣ All isolates were found to be lactose fermenters, indole positive, Voges- Proskauer negative, methly-red positive, oxidase negative, citrate negative,

urease negative and H٢S negative. Acid and gas were produced from glucose .(Table٢a) . On Kligller iron agar, yellow slope and yellow butt were observed indicating fermentation of both glucose and lactose. Also cracks due to the

.evolution of a produced gas, but not H٢S, were clearly observed :Shigella species isolates ٣٫٢٫٢ of Shigella spp was isolated in this study (Table (٪١٫٩٦) Only one strain .(٣ and Figure ١ :٣٫٢٫٢٫١Cutural characteristics On MacConkey's’s agar medium, non-lactose fermenting colonies were detected. On Salmonella Shigella (SS) agar, non-lactose fermenting colonies

.and H٢S negative colonies were detected :Microscopic Examination ٣٫٢٫٢٫٢ Non-spore forming non-capsulated Gram-negative rods were seen. :٣٫٢٫٢٫٣Biochemical reaction This isolate was indole positive, urease negative, non motile and (٢b produce acid from majority of sugar tested (Table On Kligller’s iron agar medium, red slope and yellow butt was observed, and that indicated fermentation of glucose, but not lactose. There

was no H٢S production also there was no crack on the medium indicating there was no production of gas. :Citrobactor freundii isolates ٣٫٢٫٣ and ١ of C.freundii were isolated in this study (Table (٪٥٫٨٨) Three strains .(٣ Figure :Cultural characteristics ٣٫٢٫٣٫١ On MacConkey's’s agar medium they gave pink-red colonies, and on

.SS agar gave lactose fermenting and non-H٢S production colonies :Microscopic examination ٣٫٢٫٣٫٢ Non spore-forming, non capsulated Gram-negative rods were observed. :Biochemical reaction ٣٫٢٫٣٫٣ All isolates were indole positive, methyl-red positive and produced gas from fermentation of glucose. On Kligller’s iron agar medium a yellow slope and yellow butt that indicated the fermentation of both glucose and lactose

.(were observed. The production of H٢S gas was also observed (Table٢b :Enterobacter species isolates ٣٫٢٫٤ of Enterbacter cloacae were isolated in this study (٪١٧٫٦٥) Nine strains . (٣ and Figure ١ Table) :Cultural Characteristics ٣٫٢٫٤٫١ . On MacConkey’s agar medium lactose fermenting colonies seen had irregular round edges and mucoid in nature. On SS agar yellow colonies were seen. :Microscopic examination ٣٫٢٫٤٫٢ Non-spore forming, Gram- negative short rods were seen. :Biochemical reactions ٣٫٢٫٤٫٣ The isolates were citrate positive but urease and indole negative.On Kligller’s iron agar medium, red slope and yellow butt were observed indicating the fermentation of glucose but not lactose. :Pseudomonas species isolate ٣٫٢٫٥ of Pseudomans aeruginosa was isolated in this (٪١٫٩٦) Only one strain .(٣ and Figure ١ study (Table :The cultural Characteristics ٣٫٢٫٥٫١ On MacConkey's’s agar medium, pale-coloured colonies were detected. A green pigmentation was observed. On nutrient agar the isolate gave blue On SS agar non-lactose greenish colonies were .(٨ green pigment (Figure detected. :Microscopic examination ٣٫٢٫٥٫٢ Gram-negative rod, non-spore forming and non-capsulated cells were observed. :Biochemical reaction ٣٫٢٫٥٫٣ This isolate did not ferment lactose and was oxidase positive. Pseudomonas aeruginosa produced acid from glucose, xylose and mannitol in ammonium salt sugar (ASS) medium but in peptone water sugar all sugar .(tested gave ngative reaction (Table٢b On Kligller’s iron agar, a red slope and a red butt were noticed, .(indicating neither the fermentation of glucose nor lactose (Table٢a :Proteus species isolates ٣٫٢٫٦ .of Proteus species were obtained in this study (٪١٥٫٦٨) Eight strains of Proteus (٪٥٫٨٨) of Proteus merabilis and three strains (٪٩٫٨) Five strains .(٣ and Figure ١ were isolated (Table ٣ vulgaris biogroup :Cultural characteristics ٣٫٢٫٦٫١ On MacConkey's’s agar medium pale – coloured non – lactose – fermenting colonies were detected. On SS agar they gave yellow – black

center colonies indicating H٢S production and glucose fermentation. On nutrient agar, distinctive smell colonies with swarming appearance were seen. :Microscopic examination ٣٫٢٫٦٫٢ All Proteus isolates were Gram – negative rod, non - capsulated, non- spore forming cells. :Biochemical reactions ٣٫٢٫٦٫٣ All Proteus isolates were urease positive and non – lactose fermenters. They exhibited variable indole – positive activities. Proteus isolate were also different in their fermentation of different sugar and hydrolysis of gelatin .(٢b Table) On Kligller’s iron agar medium, red slope and yellow butt were observed in all isolate of Proteus, indicating the fermentation of glucose, but

.(٢b not lactose and it was accompanied by evolution of H٢S (Table :Klebsiella species isolates ٣٫٢٫٧ .of Klebsiella spp were isolated in this study (٪٢١٫٥٧) Twelve strains (٪٧٫٦٥) and four strain of K. oxytoca (٪١٣٫٧٣)Seven strain of K. pneumoniae .(٣ and Figure ١ were isolated (Table :Cultural characteristics ٣٫٢٫٧٫١ On MacConkey's’s agar medium Klebsiella spp produced large mucoid pink colonies indicating lactose fermentation and capsule formation (Figure .(٦ :Microscopic examination ٣٫٢٫٧٫٢ Non spore – forming, capsulated, Gram – negative rod were seen. :Biochemical reaction ٣٫٢٫٧٫٣ The Klebsiella isolates of this study were Voges Proskaur – positive and methyl – red negative. Kelebsiella pneumoniae was indole negative whereas, K. oxytoca was indole positive and different in utilization of some sugars. On Kligller’s iron agar medium, yellow slope and yellow butt were observed, indicating the fermentation of

both glucose and lactose. Cracks due to a gas evolution were detected but H٢S was not

.(٢a produced (Table :Salmonella species isolates ٣٫٢٫٨

of Salmonella species were isolated in this study. One (٪٣,٩) Two species of them was (Salmonella subgenus III) and another (Salmonella subgenus IV) .(٣ and Figure ١ Table) :Cultural characteristics ٣٫٢٫٨٫١ On MacConkey's agar medium Salmonella subgenus III gave moderate pink colonies, indicating lactose fermentation but Salmonella subgenus IV

On .(٥ gave pale yellow colonies, indicating non lactose fermentation (Figure SS agar Salmonella subgenus III gave pink colonies with black center but Salmonella subgenus IV gave yellow colonies with black center. :Microscopic examination ٣٫٢٫٨٫٢ Gram-negative rods, non spore forming cells were observed. :Biochemical reaction ٣٫٢٫٨٫٣

Salmonella subgenus III was found to be lactose fermenter, but salmonella subgenus IV was found to be non lactose fermenter. Salmonella

spp were indole negative, VP positive, H٢S positive, urease negative and differ in utilization of some sugar. On Kligller's iron agar Salmonella subgenus III gave yellow slope and yellow butt indicating fermentation of glucose and lactose, cracks due to gaseous evolution were detected, blackish color due to

production of H٢S was detected. Salmoella subgenous IV gave pink slope and yellow butt, indicating fermentation of glucose but not lactose, cracks due to

gas evolution were detected, blackish color due to H٢S production was .(٢b detected also (Table :Providencia alcalifacient isolates ٣٫٢٫٩

of Providencia alcalifacient were isolated in this (٪٥,٩) Three strains .(٣ and Figure ١ study (Table :Cultural characteristics ٣٫٢٫٩٫١ On MacConkey's agar pale coloured large colonies were detected. On SS agar non lactose fermenting colonies were detected :Microscopic examination ٣٫٢٫٩٫٢

Gram-negative rods, non spore forming and non capsulated cells were observed. :Biochemical reactions .٣٫٢٫٩٫٣ Providencia alcalifacien isolated did not ferment lactose but ferment maltose, sucrose and sorbitol. They were indole positive and methyl red positive and were VP negative. On Kligller iron agar red slope and yellow butt were observed indicating fermentation of glucose

but not lactose, cracks were not detected and H٢S was not produced .(٢b Table) :Serratia species isolates ٣٫٢٫١٠

.of Serratia species were isolated in this study (٪٢٧,٥) Fourteen strains

of S. marcescence (٪٩,٨) of S.polymethca and five starins (٪١٧,٧) Nine starin .(٣ and Figure ١ were isolated (Table :Cultural characteristics ٣٫٢٫١٠٫١ On MacConkey's agar S. polymethca strains gave pink moderate lactose fermenting colonies some of them were moderate mucoid colonies; on SS agar pink colonies were obtained. S. marcescence on MacConkey's agar and SS agar produced pale colored colonies.

:Microscopic examination ٣٫٢٫١٠٫٢

None spore forming, Gram-negative rod cells were seen.

:Biochemical reactions ٣٫٢٫١٠٫٣

Biochemical reactions of two species of Serratia isolates were similar except S.polymethca strains which were arabisnose, lactose and rhammnose fermenter whereas S. marcescence did not ferment those sugars. Some strain of S. polymethca were methyl red positive but all strains of S. marcescence .(٢b were methyl negative (Table

On Kligller iron agar S. marcescence strains gave red slope and yellow

butt with cracks and H٢S was not produced. S.polymethca on Kligller iron

.(٢b agar, did not produced cracks and H٢S was not produced (Table :In vitro antibacterial activity of some antibiotics ٣٫٣

In this study cup-plate method was used to examine the sensitivity of the isolated bacteria to different antibiotics are which commonly used for treatment of child diarrhoea. Standard concentrations of these antibiotics were tested. The growth inhibition zones around the cup were measured. The antibodies tested in this study were Tetracycline; Ampicillin, Gentamycin, Streptomycin, Penicillin and Nalidixic acid, and sensitivity of sixty seven

bacterial- isolates showed (٪٩٤) bacterial isolates were examined. Sixty three

isolates for (٩٢,٥)٦٢ ,growth inhibition zones of ≥ (+++) for Nalidixic acid

isolates for (٪٥٢,٢) ٣٥ ,isolates for streptomycin (٪٩١,٠) ٦١ ,Gentamycin

isolates for (٪١٣,٤)٩ isolates for Tetracycline and (٪٤٩,٥) ٣٣ ,Ampicillin Penicillin. Nalidixic acid is the most effective antibiotic and Gentamycin was

(٪١٣,٤) ٩ second drug of choice. Penicillin was least effective drug as only

.(٤ and Figure ١٠ ,٣ isolates were sensitive to Penicillin (Table :In vitro antibacterial activity of plant extract ٣٫٤ In vitro antibacterial activity of volatile extract oil of anise ٣٫٤٫١ (P.anisum) : Three different concentrations of volatile oil extracts of anise (P.anisum) were prepared to study it is effect on bacteria associated with child diarrhoea. Most bacterial isolates inhibited by the three concentrations examined, concentration, sixty-three ٪١٠ isolates were inhibited at (٪٩٥٫٥) sixty-four % ٠٫١ isolates at (٪٨٥٫١) concentration and fifty seven ٪١ isolates at (٪٩٤) concentration as ٪١٠ concentration. The most effective concentration was and ,٤،٨ isolates were sensitive to it as shown in Table (٪٩٥٫٥) sixty-four .٤ Figure ٢٨ concentration of anise volatile oil inhibited the growth of ,٪١٠ At Enterobacter (٪١٠٠) ٥,Serratia polymuthica (٪١٠٠) ٧ ,strains of E. coli(١٠٠) ٤ ,Serratia marcescence (٪١٠٠)٥ ,Klebsiella pneumoniae (٪١٠٠)٤ ,cloaceae Proteus (٪١٠٠) ٢ ,Klebsiella oxytoca (٪٦٦٫٦) ٢ ,Proteus mirabilis (٪١٠٠) Providencia (٪١٠٠) ٢ ,Citrobacter freundii (٪٥٠) ١ ,٣ vulgaris biogroup ,Salmonella subgenus III (٪١٠٠) ١ ,Shigella dysentriae (٪١٠٠) ١ ,aclalifacient (٪٠٠٫٠) ٠ ,Yersinia intermedia (٪١٠٠)١ Salmonella subgenus IV and (٪١٠٠)١ .( ٤ Pseudomonas aeruginosa (Table .strains of E(١٠٠)٢٨ concentration of anise volatile oil inhibited ٪١ At (٪١٠٠) ٤ ,Enterobacter cloaceae (٪٨٠) ٤,Serratia polymuthica (٪١٠٠) ٧ ,coli Proteus (٪١٠٠) ٤ ,Serratia marcescence (٪١٠٠) ٥ ,Klebsiella pneumoniae ,٣ Proteus vulgaris biogroup (٪١٠٠) ٢ ,Klebsiella oxytoca (٪٦٦٫٦) ٢ ,mirabilis (٪١٠٠) ١ ,Providencia aclalifacient (٪١٠٠) ٢ ,Citrobacter freundii (٪٥٠) ١ Salmonella (٪١٠٠)١ ,Salmonella subgenus III (٪١٠٠) ١ ,Shigella dysentriae Pseudomonas (٪٠٠٫٠) ٠ Yersinia intermedia strain and (٪١٠٠)١ ,subgenus IV (٤ aeruginosa (Table ٢٦ of anise volatile oil inhibited the growth of ٪٠٫١ At concentration Enterobacter (٪٤٠) Serratia polymuthica٢ (٪٧١٫٤) ٥ ,strains of E. coli (٪٩٢٫٩) ٤ ,Serratia marcescence (٪١٠٠)٥ ,Klebsiella pneumoniae (٪٧٥) ٣ ,cloaceae Proteus (٪١٠٠) ٢ ,Klebsiella oxytoca (٪٣٣٫٣) ١ ,Proteus mirabilis (٪١٠٠) Providencia (٪١٠٠) ٢ ,Citrobacter freundii (٪٥٠) ١ ,٣ vulgaris biogroup ,Salmonella subgenus III (٪١٠٠) ١ ,Shigella dysentriae (٪١٠٠) ١ ,aclalifacient (٪٠٠٫٠) ٠ Yersinia intermedia and (٪١٠٠) ١ ,Salmonella subgenus IV (٪١٠٠) ١ (٤ Pseudomonas aeruginosa (Table In vitro antibacterial activity of water extracts of Hydnora ٣٫٤٫٢ abissynica: Water extracts without heating did not give any activity against all .(٥ ,isolated bacteria exmined (Table h did not give any ٤ ٨٠ºC) for-٧٠) Water extracts with heating at .(٥ activity against all isolated bacteria examined (Table and dried (٨٠-٧٠) concentrations, water heated %١٠ and ٪٢٠ At extracts did not give any activity against all isolated bacteria exmined (Table

.(٤ and Figure ١٠ ,٧ ,٥،٦

Bacterial species isolated from child diarrhoeal samples :١ Table collected from patients in Omdurman Pediatric Teaching Hospital Bacterial species No. of samples No. of isolates Percentage examined ٪٧٠٫٦ ٣٦ ٥١ Eschrichia coli ٪١٣٫٧ ٧ ٥١ Klebsiella pneumoniae ٪٧٫٨ ٤ ٥١ Klebsiella oxytoca ٪١٧٫٧ ٩ ٥١ Serratia polymuthica ٪٩٫٨ ٥ ٥١ Serratia marcescence

٪١٧٫٧ ٩ ٥١ Enterobacter cloaceae

٪٩٫٨ ٥ ٥١ Proteus mirabilis Proteus vulgaris ٪٥٫٩ ٣ ٥١ ٣ biogroup ٪٥٫٩ ٣ ٥١ Citrobacter freundii ٪٥٫٩ ٣ ٥١ Providncia alcalifacient

٪٢ ١ ٥١ Shigella dysentriae

٪٢ ١ ٥١ Salmonella subgenus III

٪٢ ١ ٥١ Salmonella subgenus IV

٪٢ ١ ٥١ Yersinia intermedia

٪٢ ١ ٥١ Pseudomonas aeruginosa ٪١٣١,٤ ٦٧ ٥١ Total

a: Characters and biochemical reactions of bacteria isolated from child ٢ Table diarrhoeal samples collected from patient in Omdurman Pediatric Teaching Hospital Character Eschricia Yersinia Salmonella Salmonella Enterobactor Klebsiella Klebsiella coli intermedia subgenus subgenus IV coloaceae pneumonae oxytoca III Gram stain ------Shape Rod Rod Rod Rod Rod Rod Rod Motility + + + + + - - Oxidase ------O/F F F F F F F F Urease - + + - - + + Citrate - + + - + + + VP - - - + + + D MR + + + + - - D Indole + + - - - - - Gas + + + + + + + Nitrate ND ND ND ND ND ND ND Gelatinase ND ND ND ND ND ND ND KIA: Slope: R - - - + - - - Y + + + - + + + Butt: R ------Y + + + + + + +

- - - + + - - :H٢S Gas: + + + + + + + Adenitol - - - - - + + Arabinose + + + + + + + Inositol - + - + + + + Lactose + + + - + + + Maltose + + + + + + + Raffinose + + + + + + + Rhammnose + + + + + + + Salicin + + - - + + + Sorbitol + + + + + + + Sucrose + + - - + + + Xylose + + + + + + +

Character Shigella Citrobacter Serratia Serratia Providencia Proteus Proteus. Pseudomonas spp freundii marcescence polymethca alcalifacient mirlabilis vulgaris aeruginosa

Mannitol + + + + + + + O: Oxidative; F: Fermentative; D: Doubtful; ND: Not done; R: Red; Y: Yellow; KIA: Kligller iron agar; MR:Methyl red test; VP: Voges Proskaur test

Gram ------stain Shape Rod Rod Rod Rod Rod Rod Rod Rod Motility - + - - + + - + Oxidase ------+ O/F F F F F F F F O Urease - + + - + + + + Citrate - + + + + + + + VP ------MR + + - D + + + - Indole + + - - + + - - Gas - - + + - + + - Nitrate ND ND ND ND ND ND ND + Gelatina- ND ND ND ND ND - + + se KIA: Slope: R + - - - + + + + Y - + + + - - - - Butt: R ------Y + + + + + + + + - + + - + - + - :H٢S Gas: + + + + - - - - Adenitol - - - - + - - - Arabinose + + - + - - - - Inositol - - - - + - - - Lactose - + - + - - - - Maltose + + + + - - - - Raffinose + + - - - + + - Rhamm- + + - + - - - - nose Salicin + - + + - - - - Sorbitol + + + + - - - - Sucrose + D + + + - D - Xylose + - - + - + + - Mannitol + + + + - - - - b: Characters and biochemical reactions of bacteria isolated from child ٢ Table diarrhoeal samples collected from patients in Omdurman Pediatric Teatching Hospital

O: Oxidative; F: Fermentative; D: Doubtful; ND: Not done; R: Red; Y: Yellow; KIA: Kligller iron agar; MR:Methyl red test; VP: Voges Proskaur test.

Antimicrobial activity of six antibiotics against bacteria isolated :Table٣ from child diarrhoeal samples collected from patients in Omdurman Pediatric Teaching Hospital

Bacterial No. of No. of isolate inhibited(inhibition zone diameter) by antibiotic species isolate examin- Strep Nali Amp Tetra Genta Penici ٢IU ١٠µg ٢٥µg ٢٥µg µg ٣٠ ٢٥µg ed (+++)٢ (++++)٢٤ (++++)١٠ (++++)١٣ (++++)٢٤ (++++)٢٢ ٢٨ Eschrichia (++)١ (+++)٢ (+++)٢ (+++)٢ (+++)٣ (+++)٦ coli (-)٢٥ (++)١ (++)٣ (-)١٣ (-)١ (-)١ (-)١٣ (-)٤ (++++)٤ (++++)٢ (++++)٢ (++++)٤ (++++)٢ ٤ Klebsiella (+++)١ (-)٢ (+++)١ pneumoniae (-)١ (-)١ (-)٣ (++++)٢ (++++)٢ (++++)١ (++++)٣ (++++)٣ ٣ Klebsiella (-)١ (-)١ (+++)١ oxytoca (-)١ (++)١ (++++)٦ (++++)٣ (++++)٢ (++++)٦ (++++)٤ ٧ Serratia (-)٦ (+++)١ (+++)١ (-)٥ (++)١ (+++)٣ polymuthica (++)٢ (-)١

(+++)١ (++++)٤ (++++)١ (++++)٣ (++++)٣ (++++)٢ ٥ Serratia (-)٤ (-)١ (+++)١ (-)٢ (++++)٢ (++++)١ marcescence (-)٣ (-)٢ (+++)١ (++++)٤ (++++)١ (++++)١ (++++)٤ (++++)٢ ٥ Enterobacter (+++)١ (-)١ (++)١ (-)٤ (+++)١ (+++)١ cloaceae (++)٣ (-)٣ (-)٢ (++++)٣ (++++)١ (++++)٢ (++++)٤ (++++)٢ ٤ Proteus (+++)٢ (+++)١ (-)٣ (++)٢ (+++)١ mirabilis (-)٢ (-)١ (++++)٢ (++++)١ (++++)٢ (++++)٢ (++++)٢ Proteus (+++)١ ٢ (-)١ vulgaris (-)١ ٣ biogroup

…continued :٣ Table

Bacterial No. of No. of isolates inhibited(inhibition zone diametmer) by antibiotic species isolates Strep Nali Amp Tetra Genta Penici ٢IU ١٠µg ٢٥µg ٢٥µg µg ٣٠ ٢٥µg examined (++++)٢ (++++)١ (++++)١ (++++)٢ (++++)١ ٢ Citrobacter (-)٢ (-)١ (-)١ (+++)١ freundii (-)٢ (++++)١ (++++)٢ (++++)٢ (++++)٢ (++++)٢ ٢ Providncia (++)١ alcalifacient (-)١ (++++)١ (-)١ (-)١ (-)١ (+++)١ Shigella ١ dysentriae (-)١ (++++)١ (++++)١ (++++)١ (++++)١ (++++)١ ١ Salmonella subgenus III Salmonella (+++) (++++) (+++) (+++) (++++) (-) ١ subgenus IV (-)١ (++++)١ (++++)١ (++++)١ (++++)١ (++++)١ ١ Yersinia intermedia (-)١ (++++)١ (+++)١ (-)١ (-)١ (++++)١ ١ Pseudomonas aeruginosa

;mm ٥ (+) ;١٠mm :(++) :١٥mm:(+++) ;٢٠mm :(++++) (-): no inhibition zone; Strep: streptomycin; Nali: Nalidixic acid; Amp: Ampicillin; Tetra: Tetracycline; Genta: Gentamycin; Penici: Penicillin.

Antimicrobial activity of three different concentrations of :٤ Table volatile oil extracts of Pimpinella anisum against bacteria isolated from child diarrhoeal samples collected from patients in Omdurman Pediatric Hospital Bacterial species No. of No. of isolates inhibited (inhibition zone) at concentration isolates examined % ٠٫١ ٪١ ٪١٠ (++++)٧ (++++)١٥ (++++)٢٤ Eschrichia (+++)١٨ (+++)١٣ (+++)٤ coli (++)١ ٢٨ (-)١ (++++)١ (++++)٢ (+++)٣ Klebsiella (+++)٢ (+++)٢ (+++)١ ٤ pneumoniae (-)١ (+++)١ (+++١ (++++)٢ Klebsiella (++)١ (++)٢ (++)١ ٣ oxytoca (-)١ (++++)٣ (++++)٥ (++++)٦ Serratia (+++)٢ (+++)٢ (+++)١ ٧ polymuthica (++)٢ (++++)١ (++++)٣ (++++)٥ Serratia ٥ (+++)٤ (+++)٢ marcescence (++++)٢ (++++)٤ (++++)٥ Enterobacter (++)٢ (++)١ ٥ cloaceae (-)١ (++++)٢ (++++)٣ (++++)٣ Proteus ٤ (+++)٢ (+++)١ (+++)١ mirabilis (++++)١ (++++)١ (++++)١ Proteus (+++)١ (+++)١ (+++)١ ٢ vulgaris

٣ biogroup (+++)١ (++++)١ (++++)١ Citrobacter (-)١ (++)١ (++)١ ٢ freundii

(++++)١ (++++)٢ (++++)٢ Providncia (+++)١ ٢ alcalifacient

(++++)١ (+++)١ (++++)١ Shigella ١ dysentriae (++++)١ (++++)١ (+++)١ Salmonella ١ subgenus III (+++)١ (+++)١ (++++)١ Salmonella ١ subgenus IV (++++)١ (++++)١ (++++)١ Yersinia ١ intermedia (-)١ (++)١ (++)١ Pseudomonas ١ aeruginosa mm ٥ (+) ;١٠mm :(++) :١٥mm:(+++) ;٢٠mm :(++++) ; (-): no inhibition zone. Antimicrobial activity of water extract without heating of :٥ Table Hydnora abissynica against bacteria isolated from child diarrhoeal samples collected from patients in Omdurman Pediatric Teaching Hospital Bacterial species No. of isolates No. of isolates examined inhibited ٠ ٢٨ Eschrichia coli

٠ ٤ Klebsiella pneumoniae ٠ ٤ Klebsiella oxytoca

٠ ٧ Serratia polymuthica ٠ ٥ Serratia marcescence ٠ ٥ Enterobacter cloaceae

٠ ٤ Proteus mirabilis ٠ Proteus vulgaris ٢ ٣ biogroup ٠ ٢ Citrobacter freundii ٠ ٢ Providncia alcalifacient

٠ ١ Shigella dysentriae ٠ ١ Salmonella subgenus III

٠ ١ Salmonella subgenus IV ٠ ١ Yersinia intermedia ٠ Pseudomonas ١ aeruginosa

(٨٠oC-٧٠) Antimicrobial activity of water extract with heating at :٦ Table ٤h of Hydnora abissynica against bacteria isolated from child for diarrhoeal samples collected from patients in Omdurman Pediatric Teaching Hospital Bacterial species No. of isolates No. of isolates examined inhibited ٠ ٢٨ Eschrichia coli

٠ ٤ Klebsiella pneumoniae ٠ ٤ Klebsiella oxytoca

٠ ٧ Serratia polymuthica ٠ ٥ Serratia marcescence ٠ ٥ Enterobacter cloaceae

٠ ٤ Proteus mirabilis ٠ Proteus vulgaris ٢ ٣ biogroup ٠ ٢ Citrobacter freundii ٠ ٢ Providncia alcalifacient

٠ ١ Shigella dysentriae ٠ ١ Salmonella subgenus III

٠ ١ Salmonella subgenus IV ٠ ١ Yersinia intermedia ٠ Pseudomonas ١ aeruginosa

٤h and dried oC) for ٨٠-٧٠) Antimicrobial activity of heated :٧ Table concentrations ٪١٠ and ٪٢٠ water extracts of Hydnora abissynica at against bacteria isolated from child diarrhoeal samples collected from patients in Omdurman Pediatric Teaching Hospital Bacterial No. of isolates inhibited at concentration: species No. of isolates examined

٪١٠ ٪٢٠

Eschrichia ٠ ٠ ٢٨ coli Klebsiella ٠ ٠ ٤ pneumoniae Klebsiella ٠ ٠ ٣ oxytoca Serratia ٠ ٠ ٧ polymuthica Serratia ٠ ٠ ٥ marcescence Enterobacter ٠ ٠ ٥ cloaceae Proteus ٠ ٠ ٤ mirabilis ٠ ٠ Proteus ٢ vulgaris ٣ biogroup Citrobacter ٠ ٠ ٢ freundii Providncia ٠ ٠ ٢ alcalifacient Shigella ٠ ٠ ١ dysentriae Salmonella ٠ ٠ ١ subgenus III Salmonella ٠ ٠ ١ subgenus IV Yersinia ٠ ٠ ١ intermedia Pseudomonas ٠ ٠ ١ aeruginosa

volatile oil of anise ٪٠,١ and ٪١,٠ ,٪١٠ Antimicrobial activity of :٨ Table (P.anisum) against bacteria isolated from child diarrhoeal sample collected from patients in Omdurman Pediatric Teaching Hospital

Anise volatile No. of bacterial No. of sensitive No. of resistant isolates oil isolates isolates (percentage) concentration examined (percentage)

(٪٤٫٥)٤ (٪٩٥٫٥)٦٤ ٦٧ ٪١٠

(٪٦٫٠)٤ (٪٩٤)٦٣ ٦٧ ٪١

(٪٤٫٩) ١٠ (٪٨٥٫١) ٥٧ ٦٧ ٪٠٫١ Antimicrobial activity of H. abissynica water extracts against bacteria :٩ Table isolated from child diarrhoeal samples collected from patients in Omdurman Pediatric Teaching Hospital

No. of bacterial No. of sensitive No. of resistant Water extract of H. isolates isolates isolates abissynica examined (percentage) (percentage) -٧٠) Without heating (٪١٠٠) ٦٧ (٪٠٠٫٠)٠ ٦٧ (C°٨٠

(٪١٠٠) ٦٧ (٪٠٠٫٠)٠ ٦٧ With heating

With heating, then dried (٪١٠٠) ٦٧ (٪٠٠٫٠)٠ ٦٧ concentration ٪٢٠

With heating, then dried (٪١٠٠) ٦٧ (٪٠٠٫٠)٠ ٦٧ ٪١٠ and reconsituted at

concentration of volatile oil ٪١٠,Antibacterial activity of six antibiotics :Table١٠

dried water extractact of H.abissynica against bacteria ٪٢٠ of P. anisum and isolated from child diarrhoeal samples collected from patients in Omdurman Pediatric Teaching Hospital

Bacterial Percentage of isolates inhibitied by: isolates No. of isolates exami- Strep Nali Amp Tetra Gent Penic P.anisum H.abissynica ٪١٠ ٪٢٠ ٢IU ١٠µg ٢٥µg ٢٥µg ٣٠ ٢٥µg ned µg %.٠٠٫٠ %.١٠٠ ٪٠٧٫١ ٪٩٤٫٩ ٤٢٫٩ ٥٣٫٦ ٪٩٦ ٪١٠٠ Eschrichia ٢٨ coli % %

%.٠٠٫٠ %.١٠٠ ٪٠٠٫٠ ٪١٠٠ ٪٧٥ %٥٠ ٪١٠٠ ٪٧٥ Klebsiella ٤ pneumoniae %.٠٠٫٠ %.١٠٠ ٪٢٠٫٠ ٪٦٦٫٧ ٪٦٦ ٪٢٠ ٪١٠٠ ٪١٠٠ Klebsiella ٤ oxytoca %.٠٠ %.٧٥ ٪٠٠٫٠ ٪١٠٠ ٪٤٢ ٪٢٨٫٦ ٪٨٥٫٥ ٪١٠٠ Serratia ٧ polymuthica %.٠٠٫٠ %.١٠٠ ٪٢٠٫٠ ٪٨٠ ٪٤٠ ٪٦٠ ٪١٠٠ ٪٨٠ Serratia ٥ marcescence %.٠٠٫٠ %.١٠٠ ٪٠٠٫٠ ٪١٠٠ ٪٢٠ ٪٢٠ %١٠٠ ٪٤٠ Enterobacter ٥ cloaceae %.٠٠٫٠ %.١٠٠ ٪٠٠٫٠ ٪٧٥ ٪٢٥ ٪١٠٠ ٪١٠٠ ٪٧٥ Proteus ٤ mirabilis %.٠٠٫٠ %.١٠٠ ٪٥٠٫٠ ٪١٠٠ ٪٥٠ ٪١٠٠ ٪١٠٠ ٪١٠٠ Proteus ٢ vulgaris %.٠٠٫٠ %.١٠٠ ٪٠٠٫٠ ٪١٠٠ ٪٥٠ ٪٥٠ ٪١٠٠ ٪٥٠ Citrobacter ٢ freundii %.٠٠٫٠ %.١٠٠ ٪٠٠٫٠ ٪١٠٠ ٪٥٠ ٪١٠٠ ٪١٠٠ ٪١٠٠ Providncia ٢ alcalifacient %.٠٠٫٠ %.١٠٠ ٪٠٠٫٠ ٪١٠٠ ٪٠٠٫٠ ٪٠٠ ٪١٠٠ Shigella ١ dysentriae ٠٠٫٠

%.٠٠٫٠ %.١٠٠ ٪٠٠٫٠ ٪١٠٠ ٪١٠٠ ٪١٠٠ ٪١٠٠ ٪١٠٠ Salmonella ١ subgenus III %.٠٠٫٠ %.١٠٠ ٪٠٠٫٠ ٪١٠٠ ٪٠٠٫٠ ٪١٠٠ ٪٠٠٫٠ ٪١٠٠ Salmonella ١ subgenus IV %.٠٠٫٠ %.١٠٠ ٪٠٠٫٠ ٪٢٠ ٪١٠٠ ٪١٠٠ ٪١٠٠ ٪١٠٠ Yersinia ١ intermedia %.٠٠٫٠ %٠٠٫٠ ٪٠٠٫٠ ٪١٠٠ ٪٠٠٫٠ ٪٠٠٫٠ ٪٠٠ ٪١٠٠ -Pseudomona ١ s aeruginosa %.٠٠٫٠ %.٩٥٫٥ ٪١٣٫٤ ٪٩٢٫٥ ٤٩٫٣ ٥٢٫٢ ٪٩٤٫٠ ٪٩١٫٠ ٦٧ Total % %

Strep: Streptomycin; Nali: Nalidixic acid; Amp: Ampicillin;

Tetra: Tetracycline; Gent:Gentamycin; Penic:Penicillin; IU:International Unit.

80.00%

70.60% 70.00%

60.00%

50.00%

40.00%

30.00% percentage

17.70% 17.70% 20.00% 13.70% 9.80% 9.80% 7.80% 5.90% 5.90% 5.90% 10.00% 2% 2% 2% 2% 2% 0.00%

I e t i 3 i a I di e e di n a ica ia us I tria cien th n me n n a eu ace lif r mo hia col ter ge se mu u c b y y in lca r clo ol ne su a e arcescence a lla d ct p schri ini bacter t m ia p E s lla cia o a t lla r n r Proteus mirabilisb tia ra e ne ro r Klebsiella oxytocasie Y o Shige vid Cit te e b lm ro n S le a P E Serra K Salmonella Ssubgenus IV Pseudomonas aeruginosa Proteus vulgaris biogroup

Bacterial species

Bacterial species isolated from child diarrhoea samles collected :٣ Figure from patients in Omdurman Pediatric Teaching Hospital 1.2

1 94% 95.50% 92.50% 91.00%

0.8 e g ercenta

p 0.6 52.20% 49.30% Inhibition Inhibition 0.4

0.2 13.40%

0% 0 Nalidixic acid Gentamycin Streptomycin Ampicillin Tetracycline Penicillin P. anisum H. abissynica

Six antibiotics and two medicinal plants

.Antimicrobial activity of six antibiotics, P.anisum and H :٤ Figure abissynica to bacteria isolated from child diarrhoeal samples from patients in Ondurman Pediatric Teaching Hospital

Salmonella subgenus IV on MacConkey's agar :٥ Figure

Klebseilla pneumoniae on MacConkey's agar :٦ Figure

Escherichia coli on MacConkey's agar :٧ Figure

Pseudomonas aeruginosa on nutrient agar :٨ Figure

Negative control Tetracycline

Gentamycin

Pinicillin

Streptomycin

Ampicillin Nalidixic acid

,Sensitivity of Escherichia coli to six antibiotics(Nalidixtc acid : ٩ Figure Gentamycin, Streptomysin, Ampicillin, Tetracycline and Penicillin)

P.anisum (٪١٠)

P.anisum (٪١)

H.abissynica (٪٢٠) P.anisum (٪٠٫١)

Sensitivity of Escherichia coli to three concentrations of volatile oil : ١٠ Figure dried water extract of Hydnora abissynica ٪٢٠ of Pimpinella anisum and

CHAPTER FOUR

DISCUSSION .٤ Mortality due to diarrhoeal disease in children below five year is .(١٩٧٩ ,identified as a largest single cause of mortality (Mahler In this study fifty–one diarrhoel faecal samples were collected from children suffering from acute diarrhoea in Omdurman Pediatric Teaching Hospital. These samples were inoculated onto and into suitable media and .٣٧ºC and examined for presence of bacterial growth incubated at Thirty-eight specimens gave mixed growth whereas thirteen specimens of child ٪٧٤٫٥ gave one type of bacterial growth. These results showed that of these cases were (٪٢٥٫٥) diarrhoea were caused by mixed bacteria, while caused by single type of bacteria and there was no specimens free from bacterial growth, so the present study reveal that child diarrhoea is caused mainly by mixed bacteria and sometimes may be caused by one type of (١٩٧٥)and Erwa (٢٠٠٥) bacteria. The present study agree with Mohammed findings they isolated mixed bacterial growth from child diarrhoea samples collected in Khartoum state. Several agents are known to contribute to the aetiology of diarrhoea in ,such as bacteria, virus parasite and others (٢٠٠٢ ,children (Kumar and clark Enterobacteriaceae and one strain of Pseudomonas aeruginosa were isolated from cases of child diarrhoea in this study. These isolates are probably the causal agent of child diarrhoea in Omdurman area. Out of fifty–one sample examined in this study, thirty–six strains of of child diarrhoea ٪٧٠٫٦ Escherichia coli were isolated. This result reveal that in Omdurman area was caused by E.coli. The isolation percentage of E.coli from cases of diarrhoea is higher than the isolation rate obtained by of total ٪٦٠ who reported that E.coli were isolated from (٢٠٠٥) Mohammed diarrhoeal samples collected from infant in Khartoum state. The present study who reported that E.coli is the predominate (١٩٧٥) is similar to that of Erwa (١٩٩٤) bacteria associated with child diarrhoea in Khartoum state and Ismail diarrhoeal samples taken from ٪٧٦٫٣ who reported the isolation of E.coli from reported that E.coli the (١٩٨٦) children in Khartoum state. Also Sharaf predominate bacteria associated with child diarrhoea in Joba town. ,Punte and Finlay ;١٩٧٩ ,The present study also agrees with (Lambert who reported that diarrhoeal disease in children is caused by various (٢٠٠١ species of bacteria including E.coli. From the findings of this study, it can be concluded that E.coli is the prime cause of child diarrhoea in Omdurman area. .of total samples examined ٪٢٧٫٥ Serratia species were isolated from and ٪١٧٫٧ Serratia polymethca and Serratia marsescence were islated from of samples examined in this investigation respectively. This result ٪٩٫٨ who reported that Serratia spp found (٢٠٠٢) .disagree with Greenwood et al mostly in water and soil but rarely in human feaces. These Serratia spp were isolated for the first time from diarrhoeal samples collected from Sudanese children. of ٪١٧٫٤ In the present study Enterobacter colocae were isolated from and Greenwood (١٩٨٠) .samples examined. The findings agree with Baker et al who reported that Enterobacter colocae are found in intestinal (٢٠٠٢) .et al tract of the healthy person. The E.colocae isolated from intestinal tract in the present study may be part of normal flora in Sudanese children. were (٪١٣٫٧) Twelve Klebsiella strains were isolated. Seven strains who (٢٠٠٥) Klebsiella pneumoniae. This result agrees with Mohammed (٧٫٥١) isolated K. pneumoniae from infantile diarrhoea in Khartoum state. Four (١٩٨٩) K. oxytoca strains were isolated in the present study. Cheesbrough stated that Klebsiella species are found in the normal intestinal tract of human. reported that Klebsiella spp are widely distributed in (١٩٩٧) .Koneman el al reported that(١٩٩٣)nature and gastrointestinal tract of man.. Barrow and Felam K.oxytoca has been isolated from faeces of healthy person. Klebsiella spp isolated in present investigation may be also member of normal flora in intestinal tract of Sudanese children. This study reported first time the isolation of K.oxytoca from child diarrhoeal samples in Sudan of the total ٪٥٫٩ Three strains of Citrobacter freundii were isolated from faecal samples examined in this study. These results agree with Mohammed of total isolates from infantile diarrhoea in % ٥٫١ who reported that (٢٠٠٥) Khartoum state were C.freundii. of the ٪٥٫٩ Three strain of Providencia alcalifacient were isolated from total faecal samples examined in this study. This result agrees with who stated that Providencia spp found as normal habitat (١٩٨٩) Cheesbrough in intestinal tract of the human. of total samples ٪٣٫٩ Two Salmonella species were isolated from examined in the present study. One of them was Salmonella subgenus III. This who reported the isolation of Salmonella (١٩٧٦) .finding agree with Keren et al subgenus III from patient suffering from gastroenteritis in the many age groups. The other strain of Salmonella isolated in this study was Salmonella reported that many Salmonella (٢٠٠٢) .subgenus IV. Greenwood et al serotypes affect human and Salmonella IV caused digestive tract infection in man. This investigation reported for the first time the isolation of Salmonella subgenus III and Salmonella subgenus IV in Sudan. The present study revealed the isolation of one strain of Shigella spp ,from the child diarrhoeal samples. These results disagree with Erwa (٪٢) they isolated ,(٢٠٠٥) and Mohammed (١٩٨٦) Sharaf ,(١٩٧١).Erwa et al ,(١٩٧٥) high percentage of Shigella spp from faecal samples collected from cases of child diarrhoea in Sudan. of the total samples ٪٢ Pseudomonas aeruginosa was isolated from examined in this investigation. This result confirms the result obtained by who isolated Pseudomonas aeruginosa from the infantile (٢٠٠٥) Mohammed diarrhoeal samples in Khartoum state. of total ٪٢ In this study Yersinia intermedia was isolated from diarrhoeal samples examined. During this investigation Yersinia intermedia was isolated for the first time from child diarrhoeal samples in Sudan.. The result of sensitivity testing of isolates found in this study to different of isolates ٪١٣٫٤ and ٪٤٩٫٣ ٪٥٢٫٢ ٪٩١٫٠ ,٪٩٢٫٥ ,٪٩٤٫٠ antibiotics showed that were sensitive to Nalidixic acid, Gentamycin, Streptomycin, Ampiciilin, Tetracycline and Penicillin respectively. These results agree with Mohammed who reported that child diarrhoeagenic bacteria in Khartoum state (٢٠٠٥) response to Nalidixic acid and Gentamycin but multi- resistant to Ampicillin who (١٩٧٥) and Streptomycin. Also this result is similar to that of Erwa suggested that diarrhoeagenic bacteria isolated from Sudanese children were resistant to Penicillin. From the finding of the present study it can be concluded that Nalidixic acid is drug of choice for treatment child diarrhoea in Omdurman area, because ,isolates tested ٪٩٤ it the most effective antibiotic and inhibited the growth of while Gentamycin is the second drug of choice and inhibited the growth of of isolates tested, but Penicillin is the least effective and inhibited the٪٩٢٫٥ of isolates tested, and it is not recommended for (٪١٣٫٤) ٩ growth of only treatment of child diarrhoea. of isolated bacteria were ٪٨٥٫١ ٪٩٤٫٠ ,٪٩٥٫٥ This study showed that concentration of P.anisum essential oil ٪٠٫١ and ٪١٫٠ ,٪١٠ inhibited at of bacteria associated with ٪٩٥٫٥ respectively. In the present investigation of P.anisum volatile oil as ٪١٠ child diarrhoea were inhibited at concentration who reported (٢٠٠١) .This result agrees with Larhsini et al .٥ shown in table that P.anisum gave good result against E.coli and K..pneumoniae and agrees who showned volatile oil of P.ansum gave (٢٠٠١) with the result of Elgayyar mm inhibition zone against E.coli and P. aeruginosa. The present study also ٢٥ who reported that volatile oil of P. anisum gave (١٩٩٤) .agrees with Afifi et al powerful antibacterial activity against all bacteria tested except S. typhimurium. who examined (٢٠٠٦) The result of the present study also agree with Elebead traditional medicine, and he reported that P.anisum is very active in control of child diarrhoea in Sudan. This study reveal that the best concentration of P.anisum for treatment of because this concentration inhibited the growth of ٪١٠ child diarrhoea is .of the bacterial isolated from cases of the child diarrhoea ٪٩٥٫٥ h of Hydnora ٤ C for°٨٠ – ٧٠ Water extracts with or without heating at abissynica did not reveal any activity against all bacteria examined. These who reported that water extract of (٢٠٠١) .results disagree with Elegami et al H.abissynica gave activity against Escherichia coli and Pseudomonas aeruginosa, they examined lyophilized water extract while in this experiment air dried water extract was used. The present study disagrees also with Elebead who communicated that H.abissynica is good for treatment of child (٢٠٠٦) diarrhoea. Petroleum ether extraction of H.abissnynica did not give any oil in the present study, there is no previous study reported petroleum ether extraction of H.abissnynica. The present study showed that H. abissynica is not suitable for treatment of child diarrhoea. The present study compared between the antimicrobial activities of six antibiotics with volatile oil of anise (P.anisum) and water extracts of H.abyssinica by cup-plate method and revealed that antimicrobial activity concentration of anise is equally effective as Nalidixic acid as anise٪١٠ ,of the isolated bacteria examined while ٪٩٥٫٥ inhibited the growth of .of the isolated bacteria examined ٪٩٤٫٠ Nalidixic acid inhibited the growth of ٪١٠ The present study show that using of anise volatile oil at concentration for treatment of child diarrhoea is better than Gentamycin and (٤٩٫٣) Tetracycline ,(٪٥٢٫٢) Ampicillin,(٪٩١٫٠) Sreptomycin ,(٪٩٢٫٥) for treatment of child diarrahoea in Omdurman area. Hence (٪١٣٫٤) Penicillin anise is promising drug which could be used to overcome the emerging antibiotic drug-resistant problem. CONCLUSIONS AND RECOMMENDATIONS Conclusions It can be concluding that: The bacteria associated with child diarrhoea in Omdurman area (١) include; Escherichia coli, Enterobacter cloaceae, Klebsiella pneumoniae, Serratia polymuthica , Proteus vulgaris biogroup Proteus mirabilis, Citrobacter freundii, Salmonella ,٣ subgenus III, Salmonella subgenus IV, Yersinia intermedia, Providncia alcalifacient, Klebsiella oxytoca,, Shigella dysentriae, Serratia marcescence, and Pseudomonas aeruginosa . Pseudomonas aeruginosa was the only bacterium isolated in this study from child diarrhoea that does not belonged to Enterobacteriaceae family. ,Serratia polymuthica, Serratia marcescence, Klebsiella oxytoca (٢) Salmonella subgenus III, Salmonella subgenus IV and Yersinia intermedia were isolated for first time from child diarrhoea in Sudan. The bacteria isolated from faecal sample of children with (٣) diarrhoea revealed high resistant to some antibiotics used. Nalidixic acid was the most effective antibiotic as inhibited the isolated bacteria. Gentamycin is the second (٪٩٤٫٠) growth of of the isolates were sensitive to ٪٩٢٫٥ drug of choice as it.Streptomycin is the third drug of choice which inhibit .of the tested bacteria٪٩١٫٠ Volatile oil of anise (Pimipnella anisum) could be used for treatment (٤) of isolates were sensitive to it. This ٪٩٥٫٥ of child diarrhoea as indicating it is more effective than all antibiotics examined. Water extracts of Hydnora abissynica with or without heating had no (٥) antibacterial activity against any isolates tested in this investigation. Hydnora abissynica did not give any oil by petroleum ether extract. Recommendations: Further detailed work should be performed to investigate the bacterial (١) etiology of child diarrhoea in Khartoum and other states. More antibiotic should be tested to overcome possible emergence of (٢) antibiotic drug resistance. 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