Prevalence of multi-drug resistant Salmonella Typhi in street foods in Dasmarias, Cavite

Presented by: John Royce Gnilo Mark Sumague

Typhoid Fever
Affects 20 million people

Water or food-borne disease

Caused by Salmonella Typhi Endemic in poor countries

Cases of typhoid in the Philippines

Number of Cases

2005

2006

2007

2008

2009

Fig. 1 Distribution of typhoid cases by year Philippines, 2005-2009

Fig 2. Infected areas in the Philippines

Numbe r of Cases

Fig. 3 Distribution of typhoid cases by month Philippines

Sources
Foods can be contaminated by bacteria due to improper sanitary procedures

Meat

egg

Poultry

fish products (Rheinlander et al. 2008)

Prevention and Treatment

Sanitary measures

Personal hygiene
Antibiotic therapy

Drug resistant
Resistant to Ampicillin, Chloramphenicol and

cotrimoxazole (Akinyemi et al. 2004).

Resistant occurs because they may

accumulate many genes Each coding for resistance to a single drug, within the cell (Nikaido 2009).

Objective
Determine the occurrence of multi-drug resistant

Salmonella Typhi in street foods in Dasmarias, Cavite

Statement of the problem

What is the prevalence of Salmonella in selected street foods and food stalls in Dasmarias Cavite? 2. Are culturable and non-culturable Salmonella Typhi present in selected street foods? 3. Is there a difference between the culturable and non culturable Salmonella Typhi in relation to type of street foods? 4. Is multi-drug resistant Salmonella Typhi present in street foods?
1.

Scope and limitations

Scope: 1. Food samples 2. Detection of Salmonella Typhi in street foods 3. Determination of drug resistance 4. Molecular detection of Salmonella Typhi 5. drug resistant pattern

1. 2. 3.

Limitations
S. Typhi will only be observed stn gene Antibiotics Street foods that can only be found in 5 stalls

4.

Significance of the study

Local Government Unit (LGU)

Public
Student researchers

METHODOLOGY

Research Design
Cross sectional analysis

Total of 50 samples

Research Setting

Biological Sciences Department Research Laboratory

De La Salle Health Sciences and Institute

De La Salle University- Dasmarias

Sample Collection

Isolation of Salmonella
Homogenization broth incubate at 37C for 18 hrs Streak plating in SSA

Observation

incubate at 37C for 24 hrs

Enrichment 24 hrs Colonies Antimicrobial Susceptibility Assay

centrifuge Genomic DNA kit Enriched sample

PCR

Gel Electrophoresis

Data Gathering and Statistical Analysis

Table 1. Cross-sectional analysis
Salmonella (+,-)
stall sample replication culturable 1 1 1 2 2 1 2 3 1 2 4 1 2 5 1 2 non culturable Salmonella Typhi (+,-)

Note : Same procedure in stalls 2,3,4 and 5.

Table 2. Multi-drug resistance of the isolates

Antiobiotic: Sensitive or Resistant ( less than 14 mm ) isolates ampicillin chloramphenicol cotrimoxazole streptomycin tetracyline

Note: 30 isolates will be conducted

Statistical Analysis
ANOVA

STATA 9.0
p<0.05

END

Thank You !

Genomic DNA isolation

centrifuge for 5 min at 8,000 rpm Enriched sample

pellet

resuspended in 600 L nuclei lysis solution and will be incubated at 80C for 5 min.

Suspension will be Centrifuge for 5 min at 14,000 rpm

Cell lysate RNase treated cell lysate Vortex with 200L Mix with 3 L RNase protein precipitation and incubate at solution for 20 min 37C for 30 min to 1 and incubate on ice hr. for 5 min.

Genomic DNA isolation (cont.)

DNA will be precipitated by adding equal volume of ice-cold isopropanol Centrifuge for 5 min at 14,000rpm.

pellets will be washed twice with 70% ethanol and rehydrated with 1mL rehydration solution.

Detection of Salmonella using Salmonella-specific Primers

Salmonella enterica serotypes Typhi primer A fragment of 204 base pairs (bp) of the ompC

gene will be selected fragment of 738 bp viaB gene, coding for bacterial capsule protein synthesis (Vi antigen) will be selected.

Continuation
F 5' CAC GCA CCA TCA TTT CAC CG 3';

R 5' AAC AGG CTG TAG CGA TTT AGG 3

10 pmol of forward primer and 10 pmol of reverse

primer will be used for the PCR

Mix of reagents
3.0 mM of MgCl2 final 2.0 mM dinucleotide,

10x buffer in the final concentration of 1x, 2U of

Taq polymerase, 2L of DNA as a template distilled sterile water for completing 50 L of reaction

Thermocycler
Program with initial denaturation of 95C for 2

min, then 30 cycles Each one with denaturation at 95C for 2 min, 57C as annealing temperature and 72C for 2.5 min as elongation temperate

fluid extension of 72C for 2 min

samples will be maintained at 4C until their

withdrawal.

Gel Electrophoresis
PCR amplification reactions 1.5% w/v agarose gels 100 bp marker will be included in every gel and run in TAE buffer (40mM Tris, 20mM acetic acid, and 1mM EDTA, pH 2.0)

Stain with ethidium bromide (1.0 g/L) and analysed using UVP DigiDoc-IT Imaging system.

Antibiotic susceptibility assay of Salmonella spp.

centrifuged at 3,000 rpm for 1 min.

pellet will be resuspended in sterile Phosphate Buffer Solution (PBS).

24 hrs.

MHA plate

Antibiotic discs
ampicillin (10 g)

chloramphenicol (30 g)
cotrimoxazole (30g) streptomycin (10g) tetracycline (30g)

zones of inhibition will be measured and interpreted using Clinical Laboratory and Standards Institute recommendations

incubate at 37C for 18 hrs