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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 1 10/17/2015 Key words MDRO – multidrug resistant organism MRCONS – methicillin resistant coagulase negative 2 staphylococci MRSA – methicillin resistant Staphylococcus aureus MSCONS– methicillin sensitive coagulase negative staphylococci MSSA – methicillin sensitive S.aureus OD – optical density. 10/17/2015 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 . 3 10/17/2015 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. 4 10/17/2015 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 5 10/17/2015 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. 6 10/17/2015 Results Table1:characteristics of Diabetic and non-diabetic individuals 7 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 17 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%) 10/17/2015 •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) 8 Fig.1: Types of different bacteria in Diabetic and Non 10/17/2015 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) . 9 Fig.2: Different types of bacteria in diabetic foot ulcer & age group 10/17/2015 10 Fig.3: Occurrence of different types of bacteria in diabetic foot ulcer samples from male and female individuals 10/17/2015 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 11 10/17/2015 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 12 10/17/2015 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 13 10/17/2015 14 10/17/2015 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 15 10/17/2015 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. 16 10/17/2015 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 17 10/17/2015 Gram-negative bacteria to be more prevalent than 18 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. . 10/17/2015 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. 19 10/17/2015 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 20 10/17/2015 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 21 10/17/2015