Isolation of Antibiotic resistant gene from E.coli & trial of different

antibiotics on enzyme synthesized by it for antibiotic resistance.

Introduction of E.coli

Escherichia coli commonly abbreviated E. coli is a Gram-

negative, facultative anaerobic, rod-shaped bacterium that is

commonly found in the lower intestine of warm-blooded organisms

(endotherms).

Most E. coli strains are harmless, but some serotypes can cause

serious food poisoning in their hosts, and are occasionally

responsible for product recalls due to food contamination.

The harmless strains are part of the normal flora of the gut, and can

benefit their hosts by producing vitamin K2, and by preventing the

establishment of pathogenic bacteria within the intestine.

Introduction of UTI (Urinary Tract

Infection)

Urinary tract infection has become the most common disease

encountered in clinical practice with an estimated 150 Million UTIs

occurring annually.

Although UTIs are encountered in both sexes & all ages, but it is

commoner in females. Out of every two females one has suffered in

UTIs at least once in her life.

Although etiology of UTIs is diverse encompassing both gram

positive & gram negative organisms, E.coli remains the leading

cause of 70% to 90% of the cases.

Antibiotics are the main weapons against the bacterial infections.

All the pathogenic bacteria's developing resistance to the commonly

prescribed antibiotics.

This problem is more marked in uropathogens especially E.coli

causing difficulties in treatment.

Cont.……. Community strains of E.coli are gradually showing increased

resistance towards commonly used drugs like

Ampicillin/Amoxicillin(60%) & Co- trimoxazole (10-30%).

As resistance to the drugs developed flourquinolones emerged as

the alternative therapy.

Currently the majority of urinary isolates of E.coli remain susceptible

although the resistance is on the rise since first reported in 1996s.

As the problem of antibiotic resistance increases the number of drug

curing disease decreases.

Second main cause of UTI is Klebsiella pneumoniae.

Antibiotic Resistance & Causes Antibiotic resistance is the ability of a microorganism to withstand

the effects of an antibiotics.

It is the main cause of failure in the treatment of infectious diseases

that results in increased morbidity, mortality & cost of health care.

Understanding the molecular mechanism by which resistance genes

are acquired or transmitted may contribute to the creation of new

antimicrobial strategies.

There is a large reservoir of resistant genes in the bacterial

genomes & in extra-chromosomal pieces of DNA that encodes

different mechanism of drug resistance.

Although the mechanism by which organism acquire resistance are

often well understood.

Indiscriminate use of antimicrobial by health care providers or by

way self prescribing & over the counter availability are the major

risk factors for the development of high levels of antimicrobial

resistance.

Conti…..

Some other causes are

Incorrect diagnosis,

Unnecessary prescriptions,

Improper use of antibiotics by patients.

Spreading of antibiotic resistance in

microbes Microbes undergoes mutation of genes which can spread from cell

to cell by mobile genetic elements such as plasmid, transposons, &

bacteriophages.

Plasmid is the extra- chromosomal autonomous DNA that may

encode products that may aid in virulence, pathogenicity and spread

of resistance among a wide range of bacteria.

The drug resistance character is most often encoded on plasmid.

Plasmid may effect bacterial virulence & antibiotic resistance.

Material

& Method

I. Bacterial Isolate E.coli samples are isolated form the patients having symptoms of

UTIs.

The organisms are identified using standard microbiological

techniques.

The samples are inoculated on Cysteine lactose electrolyte

deficient(CLED) media & incubated at 37 degree Celsius for 24 Hrs.

Pure strains can also be can also be obtained by sub culturing on

MacConkey agar & incubating at 37 degree Celsius for 24 Hrs.

II. Antibiotic susceptibility test

Antibiotic susceptibility pattern of E.coli can be established by

Kirby-bauer disk diffusion method.

Different antibiotic discs are placed and incubated at 37 degree

Celsius for 24 Hrs.

After incubation measurement of zones of inhibitions is done.

III. Isolation of plasmid DNA

E.coli plasmid can be isolated by Ish-Horowitz and burke

method by following Steps:

Organism is grown aerobically in 5 ml of Luria broth in the test tube

at 37 degree Celsius using a test tube shaker set at 140rpm.

Overnight culture is taken in Eppendrof tube & cells are collected by

centrifugation for 7 minutes at 12,000 rpm.

Discard the supernatant.

Suspend the pellets in 100 microliter of the Ish-Horowitz and burke

solution at room temp. for 10 minutes.

200 micro liter of lysis solution is to be added & mix vigorously.

Add 150 microliter of ice cold solution/neutralizing solution with

mixing .

Mixture is centrifuged for 15 minutes at 12,000 rpm.

Conti… Precipitate the DNA & again centrifuged for 15 minutes at 12,000

rpm.

Discard the supernatant.

Dry the pellets in dryer for 45 minutes at 45 degree Celsius.

Dissolve the dried DNA in 30 microlitre tris-EDTA buffer & kept at 4

degree celsius.

Plasmid DNA is separated by horizontal electrophoresis in 1%

agarose slab gels in a Tris-borate EDTA at room temperature at 100

volts for 3 Hrs.

DNA bands can be observed.

Photograph can be taken with UV trans illuminator.

DNA fragments compared with non resistant strain DNA fragments.

Separation of DNA fragments

Detection of gene sequence by PCR

Identification of enzymes

After identification of gene sequence we will next focus

on enzymes produced by the isolated gene that are

responsible for antibiotic resistance.

Expected results

• After the identification of enzyme we will perform

some trial of some antibiotics for inhibition of

enzyme responsible for antibiotic resistance.

• It may be helpful in giving antibiotics in

combination to avoid the antibiotic resistance.

References

I. Multiple antibiotic resistance mediated by plasmids & integrons in uropathogenic

E.coli. & K.pneumoniae. Taslima tahir lina, Sabita Rezwana Rahman & Donald James

Gomes. Department of Microbiology University if Dhaka, Dhaka, 2007.

II. Prevelence of Multiple Drug Resistant E. coli in patients of UTI Registering at a

diagnostic laboratory in Lahore Pakistan. Rabia Tanvir, Rubeena Hafeez & Shahid

Hussain. Department of Microbiology & Molecular genetics, University of Punjab, Lahore Pakistan. 2012.

III. Plasmid Profiles of multi drug resistance local uropathogenic E.coli, Klebsiella

spp., Proteusspp. & Pseudomonas spp. Isolates. B.A. Adeniyi, C.C Amajoyi &

S.I.Smith. Department of pharmaceutical Microbiology Faculty of Pharmacy, University of Ibdan, Ibdan

Nigeria.2006.

IV. Molecular level studies on multiple antibiotic & serum resistance in UTI pathogens. S.M. Dharmadhikari & S.A. Peshwe. Department of Microbiology Govt. institute of science, Caves road

Aurangabad, India.2007.

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