Isolation, Identification, Serotyping and Antibiotic Susceptibility Testing of Human Salmonella in

110 Pages·2008·0.79 MB·English

Isolation, Identification, Serotyping and Antibiotic
Susceptibility Testing of Human Salmonella in
Khartoum State

A Thesis submitted in fulfillment of the requirements for The
Master Degree of Science

By
Halima Saleh Mohy-Eldin Mohamed

B.Sc. Microbiology / Chemistry, Ain shams University

Department of Botany
Faculty of Science
University of Khartoum
April 2007

i
List of Tables

Page
Table 1. Number of isolates recovered from different
hospitals in Khartoum state 37
Table 2. Results of some presumptive tests for Salmonella 39
Table 3. Results of API 20 E tests 52
Table 4. Results of serogrouping 59
Table 5. Results of serotyping of the isolates 61
Table 6. Results of secondary biochemical tests that
group the serovars into subspecies 64
Table 7. Sensitivity of the Salmonella serotypes to
different antibiotics 69
Table 8. Molecular analysis of Salmonella using three
different primer sets 73

ii
List of plates

Page
Plate 1. Growth of Salmonella on XLD and TSI media 38
Plate 2. Some characteristics of Salmonella isolates 50
Plate 3. Results of API 20 E tests 56
Plate 4. Results of Biochemical Tests 67
Plate 5. Results of Molecular analysis with three primers 76

iii
DEDICATION

To my mother's and father's souls, my sisters,

and Abd- El-Rhman with best wishes
iv
ACKNOWLEDGMENT
I would like to express my deep
gratitude and thanks to my supervisor Dr.
Adil Ali El Hussein for his guidance genuine
criticism, and help during the course of
this study, and for all that I have learnt
from him.
Also I wish to express my thanks to Dr.
Mohammed Amin Siddig, for his help, my
thanks and appreciations are extended to
Salmonella Project Group; Sara Mahmoud,
Mayha Mohammed and Hassan Haj Safi.
My Special thanks are due to The Egypt
Management Central Laboratory Specially for
Dr. Mohammed A. Al Maksoud and Dr. Sadia A.
Moutalib. My Special thanks are due to the
Public Health Agency of Canada (PHAC)
specially; Anna Muckle, Linela Cole, Betty
Wilkie and Ketna Mistry for their great help
in serotyping of the isolates.
Also my appreciations due to Dr. Adel
Farouk for his help in Molecular analysis
v
and my thanks also extended to Ustaza
Marmar A. El Rahman for her great help.
My deep thanks are due to Dr. El Bushraa
Elshikh El Nour for his encouragement during
the course of this study.
vi
ABSTRACT
One hundred thirty human faeces samples from outpatients at a number
of public hospitals within Khartoum State were investigated for the presence of
Salmonella species. Cultural, biochemical and serological tests resulted in the
recovery of 60 Salmonella isolates. API 20E tests and serotyping confirmed the
identity of forty one of the sixty isolates as Salmonella. The forty one isolates
were found to belong to twenty five different Salmonella serovars, twenty one
of which were reported for the first time in Sudan. The forty one isolates were
subjected to more specific tests (such as growth in KCN medium, growth in
methyl red indicator medium , dulcitol and salicin fermentation) to identify the
subspecies. Twenty two of the isolates were S. enterica subspecies enterica, 2
were S. enterica subspecies salamae and only one was S. enterica subspecies
Arizona. The fermentation of d- tartrate was also used to differentiate between
the serovars expressing the same antigenic formula. Antibiotic sensitivity tests
revealed that all of the Salmonella species were resistant to Penicillin, sensitive
to Ciprofloxacin, Norfloxacin and Chloramphenicol and were variable to
Streptomycin and Gentamycin. Three primers sets were examined for their
specificity with pure cultures of the isolated Salmonella serovars. These were
16SrDNA, hilA (a pathogenicity island I virulence gene) and invasion gene
invA. The inA primer was the best of the three, it yielded PCR products with 18
of the isolates followed by 16SrDNA (16) and hilA (11).
vii
ﺺﺨﻠﻤﻟا
تﺎﻴﻔﺸﺘﺴﻤﻟا ﻰﻠﻋ ﻦﻳددﺮﺘﻤ ﻟا ﻦﻣ ﻰﺿﺮﻤﻟ زاﺮﺑ ﺔﻨﻴﻋ 130 رﺎﺒﺘﺧأو ﺺﺤﻓ ﻢﺗ
ﻢ(cid:1127)(cid:1127)ﺗ تارﺎ(cid:1127)(cid:1127)ﺒﺘﺧﻹا ﻩﺬ(cid:1127)(cid:1127)ﻬﻟ ﺔ(cid:1127)(cid:1127)ﺠﻴﺘﻧ . ﻼﻴﻧﻮﻤﻟﺎﺴ(cid:1127)(cid:1127)ﻟا ﺎ(cid:1127)(cid:1127)ﻳﺮﺘﻜﺑ دﻮ(cid:1127)(cid:1127)ﺟﻮﻟ مﻮ(cid:1127)(cid:1127)ﻃﺮﺨﻟا ﺔ(cid:1127)(cid:1127)ﻳﻻﻮﺑ ﺔ(cid:1127)(cid:1127)ﻔﻠﺘﺨﻤﻟا
تارﺎ(cid:1127)(cid:1127)ﺒﺘﺧﻹا .ﻼﻴﻧﻮﻤﻟﺎﺴ(cid:1127)(cid:1127)ﻟا ﺲﻨ (cid:1127)(cid:1127)ﺠﻟ ﻲ(cid:1127)(cid:1127)ﻤﺘﻨﺗ ﺔ(cid:1127)(cid:1127)ﻟوﺰﻌﻤﻟا تﻻﻼﺴ(cid:1127)(cid:1127)ﻟا ﻦ(cid:1127)(cid:1127)ﻣ 60 نأ ضاﺮ(cid:1127)(cid:1127)ﺘﻓإ
ﺲﻨ(cid:1127)(cid:1127)ﺠﻟ ﻊ(cid:1127)(cid:1127)ﺒﺘﺗ تﻻﺰ(cid:1127)(cid:1127)ﻌﻟا ﻩﺬ(cid:1127)(cid:1127)ه ﻦ(cid:1127)(cid:1127)ﻣ 41 تﺪ(cid:1127)(cid:1127)آأ ﺔﻴﺟﻮﻟﺮﻴﺴ(cid:1127)(cid:1127)ﻟاو (API20E) ﺔ(cid:1127)(cid:1127)ﻴﺋﺎﻴﻤﻴآﻮﻴﺒﻟا
(serovars) ﺔﻴﺟﻮﻟﺮﻴ(cid:1127)(cid:1127)(cid:1127)ﺳ ﺔ(cid:1127)(cid:1127)(cid:1127)ﻋﻮﻤﺠﻣ 25 ﻰ(cid:1127)(cid:1127)(cid:1127)ﻟإ ﻲ(cid:1127)(cid:1127)(cid:1127)ﻤﺘﻨﺗ ﺎ(cid:1127)(cid:1127)(cid:1127)ﻬﻧأ ﺪ(cid:1127)(cid:1127)(cid:1127)ﺟو ﺚ(cid:1127)(cid:1127)(cid:1127)ﻴﺣ ﻼﻴﻧﻮﻤﻟﺎﺴ(cid:1127)(cid:1127)(cid:1127)ﻟا
ﻦ(cid:1127)(cid:1127)(cid:1127)ﻣ ﺪ(cid:1127)(cid:1127)(cid:1127)ﻳﺰﻤﻟا ءاﺮ(cid:1127)(cid:1127)(cid:1127)ﺟإ ﻢ(cid:1127)(cid:1127)(cid:1127)ﺗو . نادﻮﺴ(cid:1127)(cid:1127)(cid:1127)ﻟﺎﺑ ةﺮ(cid:1127)(cid:1127)(cid:1127)ﻣ لوﻷ ﺖﻠﺠ(cid:1127)(cid:1127)(cid:1127)ﺳ ﺎ(cid:1127)(cid:1127)(cid:1127)ﻬﻨﻣ نوﺮﺸ(cid:1127)(cid:1127)(cid:1127)ﻋو ﺪ(cid:1127)(cid:1127)(cid:1127)ﺣاوو
ﺎً(cid:1127)ﻌﺒﺗو ﻼﻴﻧﻮﻤﻟﺎﺴ(cid:1127)ﻟا ﺎﻳﺮﺘﻜﺒﻟ ﺔﻔﻠﺘﺨﻤﻟا subspecies ﻰﻟإ تﻻﺰﻌﻟا قﺎﺤﻟﻹ تارﺎﺒﺘﺧﻹا
و S. enterica subspecies enterica:ﻲ(cid:1127)(cid:1127)ه تﺎ(cid:1127)(cid:1127)ﻋﻮﻤﺠﻣ ثﻼﺜ(cid:1127)(cid:1127)ﻟ ﺖﻤﺴ(cid:1127)(cid:1127)ﻗ ﻚﻟﺬ(cid:1127)(cid:1127)ﻟ
S.enterica subspecies Arizona و S.enterica subspecies salamae
ﺔ(cid:1127)ﻔﻠﺘﺨﻤﻟا تﻻﻼﺴ(cid:1127)ﻟا ﻦﻴ(cid:1127)ﺑ ﺎًﺤ(cid:1127)ﺿاو ﺎً(cid:1127)ﻨﻳﺎﺒﺗ ﺔ(cid:1127)ﻳﻮﻴﺤﻟا تادﺎﻀ(cid:1127)ﻤﻠﻟ ﺔﻴﺳﺎﺴ(cid:1127)ﺤﻟا رﺎﺒﺘﺧإ ﺮﻬﻇا
ﻦﻴﺳﺎﺴ(cid:1127)(cid:1127)(cid:1127)ﻜﻠﻓوﺮﺒﺴﻠﻟ ﺔﺳﺎﺴ(cid:1127)(cid:1127)(cid:1127)ﺣ ﺖ(cid:1127)(cid:1127)(cid:1127)ﻧﺎآ ﻦﻴﻠﻴﺴ(cid:1127)(cid:1127)(cid:1127)ﻨﺒﻠﻟ ﺔ(cid:1127)(cid:1127)(cid:1127)ﻣوﺎﻘﻣ تﻻﺰ(cid:1127)(cid:1127)(cid:1127)ﻌﻟا ﻞ(cid:1127)(cid:1127)(cid:1127)آ ﺖ(cid:1127)(cid:1127)(cid:1127)ﻧﺎآ ﺎ(cid:1127)(cid:1127)(cid:1127)ﻤﻨﻴﺒﻓ
ﺔﺒﺴ(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﻨﻟﺎﺑ ﺎً(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﻨﻳﺎﺒﺗ تﻻﻼﺴ(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﻟا تﺮ(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﻬﻇأ ﺎ(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﻤآ .لﻮ(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﻜﻨﻴﻔﻣارﻮﻠﻜﻟا و ﻦﻴﺳﺎﺴ(cid:1127)(cid:1127)(cid:1127)(cid:1127)ﻜﻠﻓورﻮﻨﻟا,
ـ(cid:1127)(cid:1127)ﻟا ﻦ(cid:1127)(cid:1127)ﻣ ﺔ(cid:1127)(cid:1127)ﺛﻼﺛ ماﺪﺨﺘ(cid:1127)(cid:1127)ﺳﺈﺑ تﻻﻼﺴ(cid:1127)(cid:1127)ﻟا ﻞ(cid:1127)(cid:1127)آ رﺎ(cid:1127)(cid:1127)ﺒﺘﺧأ ﻢ(cid:1127)(cid:1127)ﺗ . ﻦﺴ(cid:1127)(cid:1127)ﻴﻣﺎﺘﻨﺠﻟاو ﻦﺴ(cid:1127)(cid:1127)ﻳﺎﻣﻮﺘﺑﺮﺘﺳﻼﻟ
. تﻻﻼﺴ(cid:1127)(cid:1127)ﻟا ﻦ(cid:1127)(cid:1127)ﻣ ﺔ(cid:1127)(cid:1127)ﻴﻘﻧ ع راﺰ(cid:1127)(cid:1127)ﻣ ماﺪﺨﺘ(cid:1127)(cid:1127)ﺳﺎﺑ ﻼﻴﻧﻮﻤﻟﺎﺴ(cid:1127)(cid:1127)ﻟﺎﺑ ﻒ(cid:1127)(cid:1127)ﻳﺮﻌﺘﻠﻟ ﺔ(cid:1127)(cid:1127)ﺻﺎﺨﻟا Primers
تﻻﺰ(cid:1127)(cid:1127)ﻌﻟا ﻦ(cid:1127)(cid:1127)ﻣ دﺪ(cid:1127)(cid:1127)ﻋ ﺮ(cid:1127)(cid:1127)ﺒآأ ﻰ(cid:1127)(cid:1127)ﻠﻋ فﺮ(cid:1127)(cid:1127)ﻌﺘﻟا ﻰ(cid:1127)(cid:1127)ﻠﻋ ةرﺪ(cid:1127)(cid:1127)ﻘﻤﻟا InvA primer ﺮ(cid:1127)(cid:1127)ﻬﻇأ ﺪ(cid:1127)(cid:1127)ﻗو
. hilA ﻢﺛ 16Sr DNA ﻪﻴﻠﻳ ﻼﻴﻧﻮﻤﻟﺎﺴﻟا ﻦﻣ ﺔﻨﻴﻋ 18 ﻰﻠﻋ فﺮﻌﺗ ﺚﻴﺣ

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ix
Contents

Page
List of Tables i
List of Plates ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRACT IN ARABIC vii
CHAPTER ONE:
INTRODUCTION AND LITERATURE REVIEW 1
1.1 General description of Salmonella spp. 1
1.2 Distribution of Salmonella in nature 2
1.3 Classification of Salmonella, historical background and the
present situation 2
1.4 Antigenic properties of Salmonella 5
1.5 Salmonella as a major pathogen of human 6
1.6 Diagnostic Laboratory tests for Salmonella spp. 8
1.7 Vaccination against salmonellosis 10
1.8 Effects of some physical factors on growth and survival of
Salmonella spp. 10
1.9 Isolation and identification of Salmonella species 12
1.9.1 Pre-enrichment 12
1.9.2 Enrichment 12
1.9.3 Isolation of Salmonella species on selective solid media 13
1.10 Characterization of the members of the genus Salmonella 15
1.10.1 Biochemical characterization 15