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Journal Club
17/10/2015
Characteristics of microbial drug resistance and
its
correlates in chronic diabetic foot ulcer
infections
Thokur S. Murali et al., Journal of Medical Microbiology
(2014),Vol. 63, 1377–1385 .Impact factor – 2.248
Diwan Mahmood Khan
Reg. No- 151 Jan/2015
Dept. of General surgery/ Microbiology
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Key words
 MDRO – multidrug resistant organism
 MRCONS – methicillin resistant coagulase negative
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staphylococci
MRSA – methicillin resistant Staphylococcus aureus
MSCONS– methicillin sensitive coagulase negative
staphylococci
MSSA – methicillin sensitive S.aureus
OD – optical density.
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INTRODUCTION
 Foot ulcer infection is one of the major complication
of diabetes patients its altered host immunity lead to
chronic wounds.
 The range of microbes in infected wounds lead to
amputation of limbs.
 Broad -spectrum antibiotics eventually lead to the
emergence of strains resistant to many antibiotics.
 Multidrug-resistant organisms (MDROs) are common
in patients with diabetic foot ulcers .
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 Emergence and survival of MDROs in diabetic foot
ulcers produced bio-films.
 It may render leukocytes ineffective by developing
antiphagocytic properties inside the biofilm matrix.
Objective:
1. Availability of diverse classes of antibiotics for
therapy, implemented to facilitate effective
treatment of wound infection.
2. To characterize common bacterial microbes
associated with diabetic foot ulcer infections in
semi-urban and rural settings.
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 Study design
METHODS
- Sample size : 1019 diabetic and 224 non-diabetic
- Strains obtain : 1074
- Setting: Kasturba medical college , Manipal
- Duration of the study : Dec. 2010 – Aug. 2012.
- Ethical committee approval : Prior written informed
consent was obtain from all the individuals , approved
by the Ethical Committee of Manipal University.
 Isolation of microbes
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 Antimicrobial sensitivity patterns
 Detection of biofilm formation
Statistical Analysis
 Statistical analysis by SPSS statistical software (version
16.0).
 Quantitative variables expressed as means ± S.D.
 Qualitative variables presented as frequencies.
 Other analysis :- chi square (x² test).
- Student’s t-test and
- Mann–Whitney test.
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Results
Table1:characteristics of Diabetic and non-diabetic individuals
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Diabetic
Non- diabetic
Number of samples
1019
224
Number of individual
357
104
Males
284 (80 %)
91 (88%)
Females
73 (20%)
13 (12%)
Median age
58.00 ± 10.36
52.00 ± 16.16
Age < 40
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27
Age 41 – 60
201
39
Age 61 and above
139
38
Strains obtain
940
134
Gram positive strains
353 (38%)
75 (56%)
Gram negative strains
587 (62%)
59 ( 44%)
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•Graph shows a significant difference in the frequency of
occurrence in samples from diabetic vs. non-diabetic
 Acinetobacter species (p = 0.002).
 Proteus species (p = 0.001) and Enterococci (p =
0.001)
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Fig.1: Types of different bacteria in Diabetic and Non
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diabetic foot ulcer
 Highest frequency was of staphylococcus and
Pseudomonas species in the age group of 18 – 40
years, similarly in the other age group.
 Acinetobacter species occurrence was averagely
moderate in both groups.
Age groups
• 18-40 yr(white)
• 41-60 yr (strip)
• >61 yr (black)
.
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Fig.2: Different types of bacteria in diabetic foot ulcer
& age group
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Fig.3: Occurrence of different types of bacteria in
diabetic foot ulcer samples from male and female
individuals
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 Percentage of isolates of bacteria found associated
with other bacterial isolates in diabetic foot ulcer
samples
Highest- Citrobacter, klebsiella with Pseudomonas(36%)
Lowest- MRCONS with Pseudomonas (12%)
Average – Acinetobacter with Pseudomonas(25%)
 Percentage of isolates of bacteria found associated
with other bacterial isolates in non-diabetic foot ulcer
samples
MSSA with other isolates (67%)-Highest
Acinetobacter with MSSA (50%)Average/moderate
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Antimicrobial resistance profile
 Kirby- Bauer disk diffusion (followed by CLSI guidelines)
 Result confirmed by studying MIC values using Hi comb
strips.
 Enterobacteriaceae family showed high resistance in
b-lactams and least resistance against aminoglycosides.
 90 % of strains belonging to Acinetobacter spp. were
resistant to all the b-lactams including cephalosporins,
was greater than 50 %.
 Most effective drug was Netillin - 57% resistance in
diabetes and 25 % resistance in non-diabetes
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 Pseudomonas aeruginosa in diabetic patients,
piperacillin was the most effective drug with only 15 %
of strains showing resistance.
 Amikacin was effective against 70 % of Pseudomonas
aeruginosa strains
 Enterobacteriaceae family and staphylococci showed
high resistance to ampicillin (90 %).
 But enterococci were found to be more sensitive to the
same antibiotic (78 % sensitive).
 Significant difference in microbial resistance to ampicillin
for enterococci isolated from diabetic and non-diabetic
samples
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Detection of biofilm formation
 Total 355 strain obtained from diabetes and non-
diabetic patients .
 Based on tissue culture plate assay, strains were
classified as –High(O.D >0.40),
Moderate((O.D between 0.20-0.40),
Low(O.D <0.20).
 Out of 355 isolates- 159(ability to form high to
moderate levels of biofilm).
- 94 strains high biofilm producers
- 65 strains moderate biofilm producers
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 Remaining 196 strains considered- non/weak biofilm
producers.
 It was found most of the biofilm forming strains were
multi drug resistance .
 Pseudomonas aeruginosa , high ability to form high
amounts of biofilm (51 strains tested).
 23 strains of Acinetobacter were found to be
multidrug resistant, 9 strains were found to be high
biofilm producer.
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Discussion
 Diabetic
individuals tend to develop several
complications such as neuropathy and peripheral
arterial disease that contribute to foot ulceration.
 This study attempted to characterize diabetic foot
ulcer infections using conventional microbiological
and
biochemical methods
of isolation and
identification of microbes.
 In microbiological analysis of samples from diabetic
foot ulcer and non-diabetic foot wounds , total of
1074 bacterial isolates from 461 individuals
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 Gram-negative bacteria to be more prevalent than
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Gram-positive bacteria.
Gram-positive bacteria to be predominant in the
control samples(non-diabetic).
It was found from the study that diabetic patient more
case of polymicrobial infection than non-diabetic –
monomicrobial infection.
In the wound healing process-antibiotic resistance
profile of microbes.
B-lactams were not effective against member of
staphylococcus, Enterobacteriaceae family and
Acinetobacter.
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 Ciprofloxacin was considered most effective drug but
in this study 46% showed resistance to diabetic
wounds.
 But 3rd generation(ceftazidime) was effective for 41%
strains of diabetic samples and 38% in non-diabetic.
 In biofilm forming capacity of microbes associated
with diabetic foot ulcer, it was found high to
moderate biofilm forming multidrug resistance
organism.
 This finally alters the chronicity of the wound.
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 In this study it was found variation in neutrophil levels
in diabetic compared to control(non-diabetic) play
significant role in delaying wound healing.
 Researcher recommends further studies to elucidate
- relationship between microbial species
- biofilm forming capabilities and their antibiotic
sensitivities.
Conclusion:
Researcher recommends further study on greater
understanding of the effect of microbes on wound
healing and the critical host factors is overcoming in
diabetic wounds
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Research Fund
The study was supported by TIFAC – CORE in
Pharmacogenomics, Indo–Australia Biotechnology
Fund, Department of Biotechnology, the Government
of India and Manipal University, India.
THANK YOU
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