Emergence of ndm1 resistance assessment of colistin sparing antibiotic combinations

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Information about Emergence of ndm1 resistance assessment of colistin sparing antibiotic...

Published on October 17, 2020

Author: MeherRizvi

Source: slideshare.net

1. Alarming emergence of blaNDM- 1 in Gram negative bacilli in a Tertiary Care Hospital in Aligarh, Uttar Pradesh, India. Huma Naim, Meher Rizvi, Mohd Azam, Indu Shukla, Haris M. Khan Emergence of NDM -1 resistance Assessment of colistin sparing antibiotic combinations Meher Rizvi Associate Professor Department of Microbiology Jawaharlal Nehru Medical College Aligarh Muslim University

2. Alarming emergence of blaNDM- 1 in Gram negative bacilli in a Tertiary Care Hospital in Aligarh, Uttar Pradesh, India. Huma Naim, Meher Rizvi, Mohd Azam, Indu Shukla, Haris M. Khan Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh INDIA sabah al noor

3. Wonder story of 20th Century:- Miraculous Antibiotics Horror story of 21st Century:- Emergence of Superbugs Post-antibiotic era:- Stark reality: 2030

4. 2009 2010 2011

5. Carbapenem sparing protocols

6. Bacteria of concern: Gram -ve bacilli • Enterobacteriaceae • Pseudomonas species • Acinetobacter species Wide spread emergence of Metallo- beta-lactamases Premature dawn of the post antibiotic era Novel New Delhi metallo-beta-lactamase-1 (NDM-1), was discovered in K. pneumoniae in a Swedish patient after treatment in a hospital in New Delhi, India.

7. Alarming emergence of blaNDM- 1 in Gram negative bacilli in a Tertiary Care Hospital in Aligarh, Uttar Pradesh, India. Huma Naim, Meher Rizvi, Mohd Azam, Indu Shukla, Haris M. Khan Emergence of NDM -1 resistance Assessment of colistin sparing antibiotic combinations Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh INDIA

8. Aims & Objectives • Assess prevalence of Metallo beta-lactamases and New Delhi Metallo beta-lactamase -1 in carbapenem resistant Gram negative bacilli (CRGNB) in the wards of J.N. Medical College & Hospital phenotypically & genotypically • Sequence NDM1 • Explore colistin sparing synergistic combinations for management of MBL producing Gram negative bacilli

9. Material and Methods Samples collected with proper aseptic techniques Approval obtained from Institutional Ethics Committee of J.N. Medical College. Transported within an hour of collection. Organisms identified on the basis of standard guidelines. Antibiotic susceptibilities were tested as per CLSI 2014, M100-S24 guidelines

10. Antimicrobials tested Cephalosporins, Fluoroquinolones, Aminoglycosides, Nitrofurantoin, Beta lactam- BL inhibitors, Monobactam, Carbapenems, Tigecyclines, Polymyxins, Fosfomycin

11. First line drugs: Amikacin (30µg), gentamicin (10µg) Levofloxacin (5 µg), Ofloxacin (5 µg) Ceftriaxone (30µ g), cefoperazone (75µg), Cefotaxime (30µg), cefixime (30µg) cefepime (30µg), ceftazidine (30µg), cepodoxime (30µg) Ceftazidine-clavulanic acid (30/10 µg), ceftriaxone–sulbactam (30/15µg) cefotaxime- clavulanic acid (30/10 µg), cefoperazone - sulbactam. (75/75µg) Second line drugs: Ceftazidine-tazobactam (80/10µg), piperacillin (100µg), piperacillin + tazobactam (100/10µg), tobramycin (30µg), sparfloxacin (5µg), Aztreonam Third line drugs: CARBAPENEMS :Ertapenem (10µg), faropenem (5µg), meropenem (10 µg), imipenem (10µg) Fourth line drugs: Tigecycline (15 µg) Fosfomycin (200µg), Polymixin B (300 units),Colistin(10µg) T W E N T Y S I X LOST!

12. Consecutive, non duplicate CRGNB were screened phenotypically for detection of MBL producers Genotypic confirmation of MBL production Detection of MBLs

13. Phenotypic detection of MBLs ➢ Modified Hodge Test, ➢ Imipenem-EDTA Double Disk Synergy test ➢ Imipenem- EDTA Combined Disk synergy Test Lee K, Lim YS, Yong D, Yum JH, Chong Y. Evaluation of the Hodge Test and the Imipenem-EDTA Double-Disk Synergy Test for Differentiating Metallo- â - Lactamase-Producing Isolates of Pseudomonas spp. And Acinetobacter spp. J Clin Microbiol 2003;41:4623-6 Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem EDTA disc method for differentiation of metallo beta lactamase producing clinical isolates of Pseudomonas sp and Acinetobacter spp. J Clin Microbiol 2002;40:3798-801

14. • Detailed molecular characterization was carried out on116 phenotypically identified MBL producers. • blaNDM-1, blaIMP, bla VIM genes were detected. Genotypic detection of MBLs

15. Preparation of DNA template • Bacterial DNA template was prepared from freshly cultured bacterial strains • 2-3 colonies emulsified in 400µl of Tris-EDTA buffer pH 8.0 • Heated at 95°C for 10 minutes • Chilling at 4°C for 10 minutes • Centrifugation at 13,000 rpm for 5 minutes. • 300 µl of supernatant was pipetted out and stored at -20°C

16. PRIMERS Manoharan A, Chatterjee S, Mathai D, SARI Study Group. Detection and characterization of metallo beta lactamases producing Pseudomonas aeruginosa. Indian J Med 2015; 28: 241-4. • IMP Primers: Amplified product: 587 bp • IMP-A (5′-GAA GGY GTT TAT GTT CAT AC-3′) • IMP-B (5′-GTA MGT TTC AAG AGT GAT GC-3′) Manoharan A, Chatterjee S, Mathai D, SARI Study Group. Detection and characterization of metallo beta lactamases producing Pseudomonas aeruginosa. Indian J Med 2015; 28: 241-4. • VIM Primers: Amplified product: 382 bp VIM2004A 5′-GTT TGG TCG CAT ATC GCAAC-3′ VIM2004B 5′-AAT GCG CAG CAC CAG GAT AG-3′

17. PRIMERS • NDM-1 Primers: Amplified product: 621 bp amplicon • NDM A 5’-GGTTTGGCGATCTGGTTTTC-3’ • NDM-B 5’-CGGAATGGCTCATCACGATC-3’ Sequencing was outsourced to TRIYAT SCIENTIFIC CO. Nagpur, India for NDM1 Phylogenetic tree was constructed Nordmann P, Naas T, Poirel L. Global spread of carbapenemase producing Enterobacteriaceae. Emerg Infect Dis. 2011;17:1791–8.

18. Checkerboard microdilution synergy assay Time kill kinetic synergy assay COLISTIN SPARING PROTOCOLS

19. FIC index = FIC drug (A) + FIC drug (B) FIC drug (A) = MIC of drug A in combination with drug B MIC of drug A alone FIC drug (B) = MIC of drug B in combination drug A MIC of drug B alone FIC INTERPRETATION ≤ 0.5 Synergy ≥0.5- ≤1 Additive ≥ 1- ≤ 4 Indifference ≥ 4 Antagonism Clinical and Laboratory Standards Institute 2003.Performance standards for antimicrobial susceptibility testing: eighteenth informational supplement: Approved standards M100-S24. Clinical and Laboratory Standards Institute, Baltimore, USA (2014) Checkerboard Microdilution Assay

20. Time kill assay • Viable counts performed at 0, 2, 6 and 24 h. • Synergy : ≥ 3 log decrease in colony count by the combination compared to most active single agent. • Antagonism: >3 log increase in colony count Hayami, T. Goto, M. KawaharaActivities of B-lactams, fluoroquinolones, amikacin and fosfomycin alone and in combination against Pseudomonas Aeruginosa isolated from complicated urinary tract infections. Journal of Infection and Chemotherapy, 5 (1999), pp. 130-138

21. RESULTS

22. Antimicrobial resistance pattern of fourth line drugs in CRGNB isolated from patients 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% Fosfomycin, Tigecycline Aztreonam Colistin Polymixin B Ceftriazone plus sulbactam plus EDTA Antimicrobial Resistance 35.29% 43.10% 51.72% 9.48% 0.00% 3.44% Antimicrobial Resistance

23. Clinical conditions of IN-PATIENTS infected with CRGNB * 5.1% 6.12% 2.04% 27.55% 7.14% 1.02% 13.26% 8.16% 29.59% Road traffic accident Diseases of Respiratory tract Diabetes Surgery for G. I. Tract Surgery for G. U .Tract Surgery for cancer Infection Obstetric complications Fracture Surgery for GI TractBone/ SSTI *Carbapenem resistant Gram negative bacilli

24. Distribution of CRGNB* Citrobacter species, 24.13% Escherichia coli 22.41% Pseudomonas aeruginosa, 20.68% Serratia species, 14.65% Klebsiella species, 7.75% Acinetobacter species, 4.31% Proteus species, 3.44% Providencia species, 1.72% *Carbapenem resistant Gram negative bacilli

25. Modified Hodge test 90.51% CRGNB isolates were MBL positive Predominant MBL producers: ➢ Citrobacter species 23.80% ➢ Escherichia coli 23.80% ➢ Pseudomonas aeruginosa 21.90%. clover leaf like indentation

26. Imipenem-EDTA DDST 81.03% CRGNB isolates were MBL positive. Predominant MBL producers: Citrobacter species 23.40% Escherichia coli 23.40% Pseudomonas aeruginosa 21.27%. Potentiation Blank disk (10 ml 0.5 M EDTA)

27. IMP-EDTA CDST 75 % CRGNB isolates were MBL positive. Predominant MBL producers: Citrobacter species 26.43%, Escherichia coli 21.83% Pseudomonas aeruginosa 19.54%. Zone size enhanced by >5mm Imipenem plus 10 ml 0.5 M EDTA

28. Comparison of isolates for Modified Hodge Test (MHT), Imipenem-EDTA Double Disk Synergy Test (DDST) and Imipenem-EDTA Combined Disk Synergy Test (CDST). 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 90.00% 100.00% Confirmed Unequivocal 90.51% 9.48% 81.03% 18.96% 75.00% 25.00% MHT IMP- EDTA DDST IMP-EDTA DST

29. NDM-1 n=66 (83.54%) Escherichia coli :100% Klebsiella pneumoniae :78% Citrobacter species 50% Serratia species 47% Pseudomonas sp :41 %

30. VIM n=12 (15.18%) Pseudomonas sp :29 % Citrobacter species 8% Serratia species 5%

31. IMP n=1 Pseudomonas aeruginosa:1

32. Distribution of blaNDM-1, blaVIM-1 and blaIMP-1 in Gram negative bacilli (n=79) Citrobacter species, 60.71% Escherichia coli 100% Pseudomonas aeruginosa, 62.50% Serratia species, 52.94% Klebsiella species, 77.77% Acinetobacter species, 40.00% Proteus species, 50.00% Providencia species, 50.00% Aeromonas species, 0

33. Phenotypic test Sensitivity Specificity Modified Hodge Test 97.47% 29.73% IMP-EDTA DDST 85.53% 59.49% IMP-EDTA CDST 80.81% 78.38% Sensitivity & specificity of MHT, IMP-EDTA DDST, IMP-EDTA CDST in comparision to genotypic results

34. Risk factors in hospitalized patients for acquiring MBLs Risk factors Number of patients(%) n=79 Obesity 10(8.62) Hypertension 11(9.48) Tuberculosis 3(2.58) Diabetes 2(1.72) Malnutrition 15(12.93) Prolonged stay > 21 days 40 (51.72) Infection at remote site 2(1.72) Presence of drains, catheters, foley’s etc. 55 (70.68) Prior antibiotic treatment 39 (50.86) Duration of surgery> 2 hours 16 (13.79) Advanced age 18 (15.51)

35. Duration of stay in hospital ˂ 7 days 4.04% 7- 14 days 11.11% 14- 21 days 24.24% 22-28 days 25.25% ˃ 28 days 36.36% ˂ 7 days 7- 14 days 14- 21 days 22-28 days ˃ 28 days

36. Prevalence of NDM1, VIM, IMPs in clinical specimen MBL genes Pus n=56 (70.88%) Urine n=10 (12.65%) Fluid n=13 (16.45%) Total n=79 (%) NDM-1 46 (82.14) 10 (100) 10 (76.92) 66 (83.54) VIM 10 (17.85) 0 (0.0) 2 (15.38) 12 (15.18) IPM 0 (0.0) 0 (0.0) 1 (7.69) 1 (1.26)

37. Fluids Urine Pus 0% 10% 20% 30% 40% 50% 60% 70% 80% E.coli Klebsiella spp Citrobacter spp Pseudomonas spp Serratia spp 0% 11% 10% 12% 0% 70% 20% 0% 0% 10% 65% 57% 78% 57% 75% Pus Urine Fluids 0% 10% 20% 30% 40% 50% 60% 70% 80% E.coli Klebsiella spp Citrobacter spp Pseudomonas spp Serratia spp 65% 57% 78% 57% 75% 70% 20% 0% 0% 10%0% 11% 10% 12% 0% Pus Urine Fluids Distribution of NDM1in different clinical samples

38. Phylogenetic analysis of NDM-1 strains: Reported sequences included in this tree were obtained from NCBI data base. Study samples belonging to NDM-1 strains are marked with blue colour squares in our study population.

39. Outcome 66.17% patients improved 29.67% condition worsened 4.16% fatal outcome Increased duration of hospital stay Increased morbidity and mortality

40. Colistin Sparing Antimicrobial Combinations

41. Drug combinations Result Synergy Additive Indifference Antagonism Imipenem +Tigecycline 5 4 1 0 Imipenem +Amikacin 5 3 2 0 Imipenem+ Levofloxacin 4 4 2 0 Tigecycline+Amikacin 3 5 2 0 Tigecycline+Levofloxacin 2 5 3 0 Results of Antimicrobial Synergy - Checkerboard method

42. Drug combinations Result Synergy Additive Indifference Antagonism Imipenem +Tigecycline 6 3 1 0 Imipenem +Amikacin 5 2 3 0 Imipenem+ Levofloxacin 5 3 2 0 Tigecycline+Amikacin 4 3 3 0 Tigecycline+Levofloxacin 3 4 3 0 Result of Antimicrobial Synergy - Time Kill Assay

43. Post Antibiotic Era has dawned……. Colistin sparing combinations should be explored Multidisciplinary team approach: need of the hour CONCLUSION NDM1is the superbug to watch out for

44. Prof. I.ShuklaDr HibaDr Nidhi Dr Md. Azam Dr Huma Naim

45. Prof. Haris M. Khan Prof. Abida Malik

46. Shukran ALIGARH MUSLIM UNIVERSITY

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