For absolute precision and 90% confidence interval using the

For isolation and identification of E. coli, the sample size has been
calculated based on 23.07%11  prevalence of E. coli in SSIs with 10% desired absolute precision and 90%
confidence interval using the formula 12

 

                                               
n = z2
×  pexp ( 1 – pexp ) / d2 

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where n is required sample size, Z is 1.96,  p exp is
prevalence and d is desired absolute precision of 0.1.The
sample size comes to 68 subjects with SSI .To increase the precision, and for
convenience the sample size will be expanded to 100 subjects with SSI. Pus swab
samples will be collected from all the subjects .

 

STUDY DESIGN AND SAMPLING TECHNIQUE

This
study will have  a cross-sectional study
design with random sampling technique .

 

 

SAMPLE COLLECTION

The
subjects will be informed about the purpose of the study, its objectives ,
benefits and risks involved. Informed consent will be taken from the subjects
by making them sign the informed consent form. Swab samples will be taken after
consent is given .The site of  surgical
infection will be sterilized with normal saline to remove purulent debris. The
sterile cotton swabs will be moistened with sterile normal saline. Pus and
exudate will be collected from the wound by rolling the cotton swab over the
site of infection in a zig-zag motion . The cotton swabs will be placed in
sterile test tubes, The test tubes will be taken to the Department of
Microbiology NRS Medical College, Kolkata. 
The samples obtained will be labelled with the date of collection,
subject reference number, age and sex of the subject. the site of collection
and they will be identified and processed as per standard microbiological
methods and techniques.

ISOLATION OF E. coli FROM SWAB SAMPLES

The
swab samples will be immersed in nutrient broth. All nutrient broth test tubes will
be incubated aerobically overnight at 37 oC.  A loop full from the culture in each broth,
will be streaked on labelled MacConkey agar and incubated at 37°C for 24hrs.
Identification of E. coli will be carried out by Gram staining, colony morphology
and biochemical tests (Indole, mannitol, TSI test, H2S production
motility test etc).13

 

ANTIBIOTIC
SUSCEPTIBILITY TEST TO CIP IN  E.coli ISOLATES

Antibiotic susceptibility tests will be
done using the disk diffusion method in accordance with Clinical and Laboratory
Standard Institute (CLSI). CIP 5 microgram antibiotic sensitivity disks will be
used to obtain CIP resistant E. coli
isolates.

TEST
TO FIND OUT  THE PHENOTYPIC EVIDENCE OF
EFFLUX PUMP AS A PREDOMINANT MECHANISM OF ANTIBIOTIC RESISTANCE IN CIP
RESISTANCE in E. coli ISOLATES FROM SSIs

The  confirmed CIP resistant E. coli isolates will be inoculated into several batches of Ciprofloxacin
(CIP) 5 microgram anti-biotic sensitivity disks 
. Different concentrations of Verapamil will be added to each batch containing
E. coli isolates. A control batch
will be prepared on anti-biotic disks without addition of verapamil. The
antibiotic susceptibility to CIP will be measured using disk diffusion method
and a comparison will be made regarding the anti -biotic susceptibility to
Ciprofloxacin (by disk diffusion method)  between the control batch and the batches with
different concentrations of verapamil

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