Download ISOLATION AND MOLECULAR CHARACTERIZATION OF

ISOLATION AND MOLECULAR CHARACTERIZATION OF SALMONELLA
SPECIES FROM STOOL SAMPLES OF DIARRHOEAL PATIENTS FROM
HOSPITALS IN SOUTH-EASTERN NIGERIA.
A PhD RESEARCH PROPOSAL
BY
MOSES IKECHUKWU BENJAMIN (B.Sc., M.Sc., O.A.U, Ife.)
DEPARTMENT OF PHARMACEUTICAL MICROBIOLOGY & BIOTECHNOLOGY,
FACULTY OF PHARMACEUTICAL SCIENCES,
NNAMDI AZIKIWE UNIVERSITY,
AWKA,
NIGERIA.
BACKGROUND OF THE STUDY
Salmonellae are Gram-negative, rod-shaped, non-spore forming, and facultative anaerobic
bacteria which cause gastroenteritis characterized by nausea, vomiting and diarrhoea (CDC,
2008). Salmonella epidemics may occur among infants in pediatric wards, immunocompromised
individuals, elderly people and others. The frequency and gravity of these epidemics are affected
by hygienic conditions, malnutrition, and the excessive use of antibiotics that select for multiresistant strains (CDC, 2008). Salmonella organisms are aetiologic agents of diarrhoea and
systemic infections in humans, most commonly as secondary contaminants of food originating
from animals and the environment, usually as a consequence of subclinical infection in food
animals leading to contamination of meat, eggs, and milk or secondary contamination of fruits
and vegetables that have been fertilized or irrigated by faecal wastes (CDC, 2008).
The World Health Organization described in 1991 that diarrhoea is manifested by the passage of
liquid stools three or more times a day, continuously or episodically, for more than a month by
an individual.
Diarrhoea is one of the most important causes of illness and death all over the World,
particularly among infants and young children. It has been reported that five million people
especially children under the age of five years die of diarrhoea annually in Nigeria
(UNICEF/WHO, 2009).
Over the years, misuse of antibiotics has caused selection, emergence and dissemination of
antibiotic resistant Salmonella sp. with attendant public health and economic loss consequences
(WHO, 1998).
This study is, therefore, designed to isolate and characterize Salmonella sp. from the stool
samples of diarrhoeal patients in South-Eastern Nigeria with a view to identifying the antibiotic
resistance pattern, resistance genes and possible clones of virulence factors prevalent in this
environment.
OBJECTIVES OF THIS RESEARCH WORK
The specific objectives of the research are to
a) isolate and identify Salmonella species from the stool samples of diarrhoeal patients;
b) determine the susceptibility of the isolates to some antibiotics;
c) isolate resistance plasmid DNA among the multiple antibiotic resistant Salmonella
species; and isolate and identify the antibiotic resistance and virulence genes in the
multiple antibiotic resistant isolate using PCR amplification techniques.
METHODOLOGY
Study location: This research will be carried out in the Department of Pharmaceutical
Microbiology and Biotechnology, Nnamdi Azikiwe University, Awka, Nigeria. Diarrhoeal stool
samples will be collected from various Hospitals in South-Eastern Nigeria. Stool samples of
apparently healthy individuals without diarrhoea will be used as controls.
Collection of samples: The stool samples will be collected with clean universal sampling bottles
and immediately transported to the laboratory for bacteriological analysis. The collected stool
samples will firstly be enriched in Selenite F broth and incubated at 37 0C for 24 hours. A
loopful of each selenite F broth will further be streaked onto the surface of prepared SalmonellaShigella agar plates and incubated at 37 0C for 48 hours. Colonies displaying typical Salmonella
characteristics (smooth, small and colourless colonies with dark centre) will be picked and subcultured on freshly prepared Salmonella-Shigella agar plates so as to obtain pure colonies. Pure
colonies of Salmonella isolates will be picked from the Salmonella-Shigella agar plates and
inoculated on prepared nutrient agar slants and preserved in the refrigerator at 4 0C for further
tests.
Identification of Isolates: The isolated colonies will be identified using Bergey’s Manual of
Determinative Bacteriology. The bacteria will be primarily characterized and identified by
microscopic examination and by conventional biochemical and physiological tests. The cultures
will be examined for colony morphology, cell morphology, motility, Gram stain, sugar
fermentation tests and other biochemical tests such as catalase test, citrate utilization test, MRVP
test, urease test, oxidase test, nitrate reduction test, ornithine decarboxylase test, lysine
decarboxylase test, indole test, and hydrogen sulphide production test.
Antibiotic susceptibility test: This will be used to determine the antibiotic susceptibility pattern
of the Salmonella isolates. Antibiotic impregnated discs of known concentrations; gentamycin
(10 µg), augmentin (30 µg), ceftriazone (30 µg), nitrofurantoin (200 µg), cotrimoxazole (25 µg),
ofloxacin (5 µg), amoxycillin (25 µg), ciprofloxacin (10 µg), tetracycline (30 µg) and pefloxacin
(5 µg) will be used. Antibiotic susceptibility of the Salmonella isolates will be done using the
disc diffusion method and interpreted according to the guidelines of Clinical Laboratory
Standards Institute (CLSI, 2009).
Plasmid DNA extraction in Salmonella isolates: Representative multiple antibiotic resistant
isolates will be selected from the identified Salmonella isolates for plasmid analysis. Plasmid
DNA extraction will be carried out on the representative MAR isolates using TENS buffer (Tris
25mM, EDTA 10mM, NaOH O.1N and SDS 0.5%) and separated by 0.8% (w/v) agarose gel in
Tris- acetate-EDTA buffer containing ethidium bromide (20 ml of 50 X TAE and 6.0 μl of 10
μg/ml ethidium bromide per litre). Purified plasmids will then be electrophoresed in 0.8 % (w/v)
agarose for size estimation and pattern comparison. Plasmid DNA bands will then be visualized
by UV-transilluminator, photographed with a Polaroid camera and documented using a gel
electrophoresis documentation system. The molecular sizes of the unknown plasmid DNA will
then be estimated by comparing the distance travelled with that of the molecular weight of the
standards.
Polymerase Chain Reaction (PCR) amplification and detection of PCR products:
Representative multiple antibiotic resistant Salmonella isolates will be screened for the detection
of the BlaCTX resistance gene, GyrA resistance gene and invA virulence gene using Polymerase
Chain Reaction (PCR) amplification technique. The selection of the isolates was based on the
antibiotic reaction profiles. The primers to be used are:
BlaCTX: (BlaCTX- Forward) - ATG TGC AGY ACC AGT AAR GTK ATG GC and (BlaCTXReverse) - TGG GTR AAR TAR GTS ACC AGA AYC AGC GG will be used.
GyrA: (GyrA- Forward) - CGT TGG TGA CGT AAT CGG; (GyrA- Reverse) - CCG TAC CGT
CAT AGT TAT
InvA: (InvA-Forward) - ACA GTG CTC GTT TAC GAC CTG AAT; (InvA-Reverse)- AGA
CGA CTG GTA CTG ATC GAT AAT were used.
Statistical analysis
Statistical analysis will be performed using T-test and Pearson correlation index at P < 0.05 with
statistical software SPSS Data Editor Version 16.
EXPECTED RESULTS
At the successful completion of this research project, I expect to
1. isolate and identify Salmonella species from the stool samples of diarrhoeal patients;
2. identify the antibiotic resistance pattern of the Salmonella isolates;
3. isolate and identify antibiotic resistance genes (blaCTX and GyrA), virulence gene
(invA) and possible clones of virulence factors prevalent in this environment as this
information could help limit the spread of diarrhoea diseases and provide data about the
best choice of antibiotics for treatment.
REFERENCES
Bergey’s Manual of Systematic Bacteriology. 2B (2nd ed.). New York: Springer. pp. 1108,
ISBN 978-0-387-24144-9.British Library no. GBA561951.
Centre for Disease Control and Prevention (CDC). (2008). Public Health Laboratory
Information System (PHLIS). Salmonella surveillance, annual summary, 2006, Atlanta, GA.
http://www.cdc.gov/ncidod/dbmd/phlisdata/salmtab/2006/SalmonellaAnnualSummary2006.pdf
Clinical and Laboratory Standards Institute CLSI/NCCLS. (2009). Performance standards
for antimicrobial susceptibility testing.Nineteenth Information supplement. Vol 29, No. 3.
CLSI/NCCLS document M100-S19, Wayne, Pennsylvania.
United Nations Children’s Fund and World Health Organization (UNICEF /WHO). (2009).
Why children are still dying and what can be done. ISBN 978-92-806-4462-3 (UNICEF); ISBN
978-92-4-159841-5 (NLM classification: WS 312) (WHO).
World Health Organisation (WHO). (1998). Use of quinolones in Food Animals and Potential
Impact on Human Health.Report of a World Health Organisation’s Meeting, 2-5 June 1998,
Geneva, Switzerland.