RESEARCH GRANT PROPOSAL
02-Dec-15
1
Detection of beta-lactamase drug resistance
producing in Acinetobacter species among
diabetic foot ulcer patients attending a tertiary
care hospital
Diwan Mahmood Khan
PhD Scholar
Yenepoya University
02-Dec-15
2
AGENCY APPLYING FOR:
Indian Council of Medical Research (ICMR)
02-Dec-15
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INVESTIGATORS
P-INVESTIGATORS:
CO-INVESTIGATORS:
Dr. M.S.Moosabba
Professor and HOD
Dept of General Surgery
Yenepoya Medical College
Mangalore.
Diwan Mahmood Khan
Research Scholar
Dept. of Microbiology,
Yenepoya Medical College
Mangalore.
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INTRODUCTION:
• Diabetic foot ulcer infection, approximately 25% of
patient with diabetes was experience diabetic foot ulcers
during their lifetime.1, 2
• Diabetic foot ulcer infection is one of the major
complications of diabetes patient, approximately 57
million people nationwide will affected by the year
2025.3
• Patient with diabetic foot ulcer having 10 times greater
risk being hospitalized infection compare to without
having diabetes.4
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Continue..
• According to the severity of diabetic foot ulcer, there
are four classification system (Wagner, PEDIS,
University of Texas and SINDBAD) which are used
worldwide. 5
• The Acinetobacter species are gram negative
coccobacilli, non motile,6 catalase positive, oxidase
negative and found ubiquitously in the environment,
it is strict aerobic bacteria. 7
• It is opportunistic pathogen affecting people with
compromised immune systems, and is become
hospital acquired infection. 8
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Continue..
• There are very few reports on multi drug resistance
Acinetobacter species associated with diabetic foot
ulcers infection in India and abroad. 9,10
• Over the last three decades, interest in Acinetobacter
species has grown rapidly, due to the emergence and
outbreak of drug- resistant. 11, 12
• Acinetobacter species are intrinsically resistant to
many antimicrobial agents, 13 and resistance to blactams is most commonly cephalosporin and
carbapenem.14
• Emergence and survival of drug resistance
Acinetobacter species which lead to biofilm
formation15.
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• Resistance against beta-lactamase group of drugs,
widely used for their treatment and their efficacies
are increasingly being compromised. 16
• Resistance mechanism to carbapenems have been
reported in Acinetobacter species, i.e carbapenem
hydrolyzing-lactamases of molecular Ambler class B
(metallo enzymes) which include blaIMP and class D
enzymes oxacillinases type- OXA-23, OXA-58. 17
• Cephalosporin resistance Acinetobacter species is
rising significantly because of the over-expression
intrinsic cephalosporinase and chromosomal bla ADC
genes in Acinetobacter species can lead to overexperssion of the ADC-type b-lactamase. 18
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Review of Literature:
Indian scenarios:
• P. Ramakant et al., (2011) Lucknow, stated that
Gram-negative bacteria dominant in the diabetic foot
ulcer patient. Infection with polymicrobial drug
resistant Gram-negative bacilli is common and need
to change empirical therapy. 19
• Jain Manisha et al.,(2012),Gujarat, stated that
diabetic foot infections are more predominant gram
negative bacilli. Evaluation of severity of diabetic foot
by Wagner’s grade. 20
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• Priyadarshini Shanmugam et al.,(2013), kanchipuram,
stated that Acinetobacter species is the third
common gram negative bacteria follow to diabetic
foot infection. 4
• Thokur S et al.,(2014), Manipal, stated that
Acinetobacter species is the second common gram
negative bacteria follow to diabetic foot infection.
They produced moderate to high levels of biofilms
formation and drug resistance. 21
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International scenarios:
• J.J. Mendes et al.,(2012),Portugal, stated that
prevalence of drug resistance organisms in diabetic
foot ulcer patient were high to previous
indiscriminate antibiotic use. 22
• Abd Al-Haround amead Hefni et al.,(2012),Egypt,
stated that around 10% diabetic foot ulcer infection
caused by Acinetobacter species. This is third
common gram negative isolates in foot ulcer. 23
• Azar Hadadi et al.,(2014), Iran, stated that Gram
negative bacilli isolates in diabetic foot ulcer patient
is high and variations drug resistance of gram
negative organism according to geographical
condition. 4
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Purpose of the study:
This study aimed to detect cephalosporin and
carbapenem drug resistance gene mechanism in
Acinetobacter species from diabetic foot ulcer
patients.
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Objectives:
• To isolate the Acinetobacter spp. and detection of the
beta-lactamase drug resistance by the Kirby bauer disk
diffusion and Modified Hodge Test method from the
diabetic foot ulcer patients.
• To detect the presence of carbapenem(OXAIMP
OXA23,OXA58) and cephalosporin gene (blaADC) among
beta-lactamase resistant Acinetobacter spp. by Real time
PCR.
• To perform sequencing for the beta-lactamase resistant
Acinetobacter spp. for the possible mutation in the
carbapenem and cephalosporin gene
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METHODOLOGY:
• Research Design:
Prospective study
• Study Site:
Yenepoya Medical Hospital, Mangalore.
• Study duration: 36 months
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MATERIAL and METHOD:
• INCLUSION CRITERIA:
The isolates are included in the study, if they are
diabetic foot ulcer infection only.
• EXCLUSION CRITERIA:
The samples of non - diabetic foot ulcer infection will
be excluded.
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• Sample size: 150 numbers.
• Ethical clearance- will be obtained from the
Yenepoya University, Mangalore.
• Informed consent : will be taken in study.
• Statistical assessment using: SPSS version 20
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SAMPLE COLLECTION :
• Pus, tissue & wound Swab should be collect according to
the standard protocol.
• Samples will be process for gram-stain and culture on
blood agar and MacConkey agar media and incubate
overnight at 37◦C for 18- 24 hrs.
• Identification of Acinetobacter species isolates will be
done by standard biochemical methods. 25
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Isolation and identification:
Acinetobacter species will be identified using standard
methods :
• Gram staining
• Culture on blood agar and MacConkey agar media ,
• Biochemical identification method
PHENOTYPIC DETECTION METHOD:
Antibiotic susceptibility testing: For beta-lactam antibiotics
By Kirby-Bauer disk-diffusion method as per CLSI
guidelines.
18
02-Dec-15
Kirby Bauer Disc diffusion method :
Allow the Muller Hinton agar(MHA) plate and disk
cartriadge to come to room temperature before use.
Prepare the test inoculum and compare with the 0.5
McFarland standard .
Lawn culture the bacterial suspension evenly in 3 planes
onto the surface of the MH agar plate, using a cotton
swab. Rim the edge of the plate.
Place the antibiotics disks over the MH agar plate with the
help of sterile forcep.
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Kirby Bauer Disc diffusion method 26 :
Allow the Muller Hinton agar(MHA) plate and disk cartriadge to
come to room temperature before use.
Prepare the test inoculum and compare with the 0.5 McFarland
standard .
Lawn culture the bacterial suspension evenly in 3 planes onto
the surface of the MH agar plate, using a cotton swab. Rim the
edge of the plate.
Place the antibiotics disks over the MH agar plate with the help
of sterile forcep.
20
02-Dec-15
Contd…
press the disks to make a firm attachment between the
disks and the agar.
Incubate the MH agar plate overnight in a incubator at
35ºC.
Check the zone of inhibition and interpretate the result
of individual antibiotic disc as Sensitive (S),
Intermediate (I) and resistant (R).
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Modified Hodge Test (MHT) for detection of
Carbapenemase:
• Carbapenemase production is detected by the MHT
when the test isolate produces the enzyme and
allows growth of a carbapenem susceptible strain
towards a carbapenem disk.
• Place a 10 μg Meropenem disk in the centre of the
inoculated MHA plate. In a narrow but thick straight
line ,streak test organism from the edge of the plate.
• The MHT Positive test has a clover leaf-like
indentation & Negative test has no growth
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GENOTYPIC DETECTION METHOD :
DNA extraction:
• A single colony will be taken from blood/macconkey agar
which will be incubated overnight and emulsified into 100 μl
of phosphate buffer salt.
• After incubation for 10 min at 95°C, 50 μl of proteinase K
(100 mg/l) and 150 μl of TE (1 mM EDTA/10 mM Tris, pH
7.5) will be added to the suspension and incubated for a further
20 min at 37°C . 26
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Multiplex PCR detection:
• The multiplex PCR will be performed using the following
oligonucleotides as primers of blaADC and OXA23,OXA-58,blaIMP .
• The reaction will be performed under different condition:
0.2mM of each primer will be used.
• The following time temperature profile will be chosen:
one initial cycle of denaturation for 5 min at 94◦C and 45
cycles of 30 sec at 94◦C, 30 sec at 60 ◦C, and 30 sec of
extension at 72◦C. after the thermal cycle, the amplicons
will be electrophoresed in a 2% agarose gel and stain with
ethidium bromide . 27
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Carbapenem gene
Primer(28)
bla IMP
F:CTACCGCAGCAGAGTCTTTGC
R:GAACAACCAGTTTTGCCTTACC
OXA-23
F:GATCGGATTGGAGAACCAGA
R:ATTTCTGACCGCATTTCCAT
OXA-58
F:AAGTATTGGGGCTTGTGCTG
R:CCCCTCTGCGCTCTACATAC
Cephalosporin gene
Primer(29)
bla ADC
F:ATGCGATTTAAAAAAATTTCTTGT
R:TTATTTCTTTATTGCATTCAG
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Social Relevance
• To determine the drug resistance & control
the hospital acquired infection
• Helps the Clinician to select proper antibiotics
in diabetic foot ulcer infection.
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RESEARCH OUTCOME :
• This study will be able to determine the betalactamase drug resistance genes among
Acinetobacter species in hospital isolates from
diabetic foot ulcer.
• Early detection of hospital acquired infection causing
Acinetobacter species by Real time PCR method.
• To reduce prolong stay in hospital due to diabetic
foot ulcer infection causing Acinetobacter species
and implement wound management.
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TIMELINE
EVENTS
6 MONTHS 12
18
24
MONTHS MONTHS MONTHS
30
36
MONTHS MONTHS
Review of
Literature
Standardisation
of procedure
Research
Experiment
Publications
Writing Thesis
02-Dec-15
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BUDGET REQUIREMENTS:
Expenses
JRF– 14000/month
Consumable
AST ,MHA
First Year
168000/-
50,000/-

PCR kits
50,000

Primer and Probes
1,20,000/-

Sequencing
analysis
Non-consumable

Micropipette10 100ul)

Equipment for
PCR- plastic
ware, glassware
Contingency
Overhead Charges
Total
02-Dec-15
-
Second Year
Third
Year
Total
168000/-
168000/-
504000/-
50,000/-
-
1,00,000/-
50,000
-
1,00,000/-
1,20,000/-
-
2,40,000/-
-
2,00,000
2,00,000
50,000/-
50,000/-
5000/-
5000/-
5000/-
15,000/-
20,000/-
15,000/-
15,000/-
50,000/-
4,63000
408000
388000
12,59,000/-29
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