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Name of the candidate Name of the guide Dr. Rouchelle Tellis Dr. M.S Moosabba

Comparison of virulence factors among antibiotic sensitive and multidrug resistant clinical isolates of Pseudomonas aeruginosa - An in-vitro study. Name of the candidate Name of the guide Dr. Rouchelle Tellis Dr. M.S Moosabba

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Name of the candidate Name of the guide Dr. Rouchelle Tellis Dr. M.S Moosabba

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  1. Comparison of virulence factors among antibiotic sensitive and multidrug resistant clinical isolates of Pseudomonas aeruginosa - Anin-vitro study Name of the candidate Name of the guide Dr. Rouchelle Tellis Dr. M.S Moosabba PhD Reg No.: 061/Jan 2012 Professor & Head, Dept. of Surgery Assoc. Prof, Microbiology Yenepoya Medical College Yenepoya Medical College Name of the research Co-guide Dr. Ronald Roche Professor, Dept. of Microbiology Malabar Institute of Medical Sciences, Kannur

  2. Comments / remedial action • Introduction and background • Present status -International, national, local scenario • Need and relevance of the work • Aim, Objectives • Methodology: Sample size, inclusion & exclusion criteria • Results and conclusions • Proposed work for next 6 months & timeline • References

  3. INTRODUCTION: • P. aeruginosa : opportunistic invader, major nosocomial pathogen : in immuno-compromised, immune-deficient or surgically manipulated hosts • Infections caused: • LRTI (including VAP) • Wound infections • UTIs • BSIs & device associated infections • MULTIDRUG RESISTANCE : accelerating problem worldwide, difficult target for antibiotic therapy

  4. INTRINSIC RESISTANCE: Constitutive expression of efflux pumps & low permeability of the OM • EXTRINSIC RESISTANCE: Acquisition of plasmid mediated resistance genes (β-lactamases) • QUORUM SENSING: population density based, cell to cell communication system controlling growth regulation, expression of virulence factors & antibiotic resistance • BIOFILMS: survival advantage & protection from host immune system and antimicrobial agents

  5. Β-LACTAM RESISTANCE: • Chromosomally encoded Amp C β–lactamases • Plasmid mediated ESBLs • EFFLUX PUMPS: β–lactams and fluroquinolones. • MBL producing P.aeruginosa : often associated with outbreaks of nosocomial infections (septicemia, VAP) : carbapenem therapeutic failure

  6. Need for the study based on literature review • Antibiotic resistance was believed to come at the cost of bacterial fitness and reduced virulence. • Research by Skurniket al. challenges the common concept that resistant bacteria are less fit(Skurnik D, et al. (2013) Enhanced in vivo fitness of carbapenem resistant oprD mutants of Pseudomonas aeruginosa revealed through high-throughput sequencing. Proc NatlAcadSci USA 110:20747–20752.) • Tested this assumption in cystic fibrosis patients who are persistently colonized with antibiotic-resistant PA. Enhanced fitness is due to lack of Opr D and this is associated with acquisition of carbapenem resistance

  7. This conferred carbapenem resistance + enhanced serum bactericidal resistance with increased cyto-toxicity. • Thus, if carbapenem resistance develops during antibiotic therapy of P. aeruginosa infections, it may lead to enhanced fitness and virulence in infected hosts • FIT AND RESISTANT is a worst case scenario with bacterial pathogens.(Filloux A. PNAS 2013; vol. 110: 2036)

  8. Review of literature-  International scenario: • Paucity of literature comparing virulence factors among antibiotic sensitive and MDR P.aeruginosa. • Shalyet al demonstrated: ESBL producing K. pneumoniaeare more invasive: greater serum bactericidal resistance , cell invasion & fimbrialadhesins. • Brazilian study of 84 CAZ resistant P.aeruginosa, 37% and 4% were found to produce MBL and ESBL respectively. • NEJM Dec 11 2013: ‘Antibiotic-Resistant P.aeruginosa Can Become More Virulent’ by Richard T Ellison

  9. National scenario: • No study from India comparing the virulence factors among the antibiotic sensitive and resistant isolates of P. aeruginosa • 2011 De AS et al reported MBL production among 33% of P. aeruginosa isolated from blood and endotracheal secretions of patients in ICUs of a tertiary care hospital.

  10. Infections by MDR ESBL & MBL producing PA: often associated with adverse clinical outcomes. Not yet clear from literature if this high mortality can be related to increased virulence of these antibiotic resistant strains. • This study aims to identify and compare the possible correlation between virulence and antibiotic resistance among clinical isolates of P. aeruginosa • This could help decide the significance and treatment modality for MDR P. aeruginosa

  11. Working hypothesis: MDR clinical isolates of PA are more virulent than antibiotic sensitive isolates, giving the former better survival advantage and increased potency to cause invasive infections. Aim of the study:To compare and correlate the expression of virulence factors among antibiotic sensitive and MDR clinical isolates of P.aeruginosa. Objectives of the study: • To isolate, identify and study the antibiogram of P.aeruginosa isolatedfrom clinical samples. • To phenotypically detect ESBL, MBL and Amp C β-lactamaseproduction among cephalosporin resistant isolates

  12. Objectives……….. • To identify production virulence factors: serum bactericidal resistance, mannose sensitive and resistant pili, swimming and twitching motility, haemolysin, phospholipase C, elastase and slime production. • Compare & correlate virulence factor production among antibiotic sensitive and MDR isolates • To identify presence of IMP and VIM MBL genes among MDR isolates • To study the in-vitro effect of antibiotic combinations against MDR P.aeruginosa

  13. Materials: • Sample Source: PA isolated from clinical samples received at Microbiology laboratory YMCH from Jan 2013- Jan 2016. Sample size: Three groups of samples:

  14. Inclusion criteria: • Only one strain of P.aeruginosa per patient included Exclusion criteria: • PA isolated from urine with a colony count of <105 CFU/ml. • PAisolated from pus, without pus cells in direct gram stained smear. • PAisolated from a single blood culture when there is no clinical correlation

  15. Methods: • Collection, processing of clinical samples and identification of PA using standard tests • Susceptibility to CAZ, CPZ, aztreonam, gentamicin, amikacin, tobramycin, ciprofloxacin, netilmicin, piperacillin, pip-tazo, CPZ- sulbac, ticarcillin, imipenem, meropenem and colistin: tested & interpreted by disc diffusion method: CLSI 2013 • P. aeruginosa ATCC 27853 - quality control • For long term use bacteria stored at -700C in trypticase soy glycerol broth

  16. Test for ESBL production: CLSI phenotypic disc confirmatory test. K. pneumoniae ATCC 700603 control strain for ESBL detection • Test for inducible Amp C β-lactamase production: • Disc antagonism test (DAT): inducing agent cefoxitin disc placed adjacent to cephalosporin discs. Blunting of the cephalosporin zone adjacent to the cefoxtin disc is suggestive of AmpC β-lactamase production. • Inhibitor based Amp C detection test: using 30 μg of cefoxitin disc with 400 μg boronic acid disc

  17. TEST FOR MBL DETECTION: Imepenem-EDTA DDST by Yong D et al. • MBL GENE IDENTIFICATION: Identification of IMP & VIM genes among carbapenem resistant PA isolates by real time PCR • Virulence factor production in antibiotic sensitive isolates will be compared and correlated with virulence factor production of MDR P.aeruginosa • P. aeruginosa C-10 (VIM-2) and C-7 (IMP-7) sourced from Calgary laboratory Canada used as positive control strains for MBL production

  18. Production of the following virulence factors: • Serum bactericidal assay: According to R Podschunet al. • Hemagglutination assay: Detected by clumping of erythrocytes by fimbriae of bacteria in presence of D-mannose, (modified Siegfredet al procedure) • Haemolysin production: detected using 5% sheep blood agar. • Phopholipase C activity: Method of Haberman and Hardtusing egg-yolk agar

  19. Swimming motility:as described by Rashid & Kornberg • Twitching motility:as described by Rashid & Kornberg • Slime/alginate production:using micro-titre plate adherence method • Statistical analysis of data: Expression of virulence factors in antibiotic sensitive & resistant isolates will be compared by chi-square test using SPSS software 17.0 version • Correlation of virulence factors with the antibiotic phenotype will be tested using independent sample t-test, where p<0.05 will be considered statistically significant.

  20. Work progress from July – Dec 2015

  21. Table 1: Clinical source of P.aeruginosaisolates:

  22. NLF colonies with spreading edges obtained on Mac Conkey agar were identified by biochemical testing Table 2: Biochemical Identification of PA isolates

  23. Table 3: Antibiogram of P.aeruginosa isolates

  24. Table 3: Antibiogram of P.aeruginosa isolates

  25. Table 4: Classification based on the antibiogram pattern

  26. Table 5: Antibiotic resistance pattern of MDR-PA

  27. Table 6: Sample wise distribution of MDR P.aeruginosa

  28. TABLE 7: β-lactamase production by P.aeruginosa:

  29. Table 8: Virulence factor production among test and control groups

  30. STATISTICAL ANALYSIS: Comparison of virulence factor production by antibiotic sensitive control strains and MDR-GNB test strains by chi square test revealed that: Hemolysin production (p= 0.0038) Biofilm formation (p= 0.0042) Serum resistance (p= 0.0036) Swimming motility (p= 0.0038)

  31. Table 9: Comparison of virulence factor production among antibiotic sensitive and MDR PA from various sources

  32. Table 9: Comparison of virulence factor production among antibiotic sensitive and MDR PA from various sources

  33. Minimum Inhibitory Concentration (MIC) testing: MICs of CAZ, AMK, IMP, CIPRO were tested for 20 MDR isolates by broth micro-dilution method according to CLSI guidelines 2013 Table 10:Solvents and diluents used for the antibiotics Table 11: MIC ranges, MIC50 & MIC90 values of MDR P.aeruginosa:

  34. COMBINATION ANTIBIOTIC TESTING: Antibiotic combinations tested in-vitro against MDR PA: CAZ-AMK, CAZ-CIPRO, IMP-AMK, IMP-CIPRO by checker- board assay. Calculation and interpretation of FIC: • The FICs were calculated as follows: Σ FIC = FIC A + FIC B • FIC A : MIC of drug A in combination/MIC of drug A alone • FIC B : MIC of drug B in combination/MIC of drug B alone. • The combination is considered Synergistic -Σ FIC is ≤0.5, Indifferent: Σ FIC is >0.5 to <2 Antagonistic : Σ FIC is ≥2.

  35. Table 12: Results of combination antibiotic testing for MDR PA: (N=20) Comparing FICI of CAZ-AMK with IMP-AMK & IMP alone, p value of 0.261 suggests that the difference is not statistically significant

  36. DNA extraction: • DNA was extracted from 30 isolates each from Control groups 1 & 2 and all MBL producers in group 3 DNA extraction from bacterial cultures DNA extraction from bacterial cultures

  37. BIOFILM FORMATION: wavelength of 580 nm with a Multimode 29/01/2015 Quantitative estimation of biofilm by microtiter plate assay. OD was recorded at 580nm using microtiter plate reader (FLUOstar Omega - Bitotron Health care) The mean values of OD blank were subtracted from the mean values of OD of test strains. 22.5.2015 Biofilm detection: Micro-titre plate method optical density cut off (Odc) value= average OD of NC+ 3SD of NC

  38. MINIMUM INHIBITORY CONCENTRATION OF CAZ CHECKER BOARD ASSAY 12.06.2015 08.02.2015 MIC detection: Micro broth dilution method 11.06.2015 Antibiotic combination testing by checker board assay

  39. MBL PRODUCTION MBL DETECTION SWIMMING MOTILITY 14.3.2015

  40. PRESENTATIONS AT CONFERENCES Fit and resistant: A comparison of virulence factors among antibiotic sensitive and resistant P. aeruginosa. At IAMM-KC Feb 2014, KMC Manipal: BEST ORAL PAPER AWARD In vitro assessment of antibiotic combinations against multidrug resistant P. aeruginosa: An in-vitro study, has been accepted for oral paper presentation at 39th annual conference of IAMM at JIPMER, Nov 2015. Is it possible to reduce carbapenem dependency in treatment of MDR P.aeruginosa infections in acute care settings: a prelimnary study. At International Congress of Infectious Diseases (ICID), March 2-5th 2016, Hyderabad

  41. Publication:

  42. ICMR –STS Projects obtained • Activity of antibiotic combinations against multidrug resistant gram negative bacilli: An in-vitro study: ICMR –STS 2013 • Correlation between antibiotic resistance and virulence factors of multidrug resistant gram negative bacilli: ICMR –STS 2014

  43. Proposed work for next 6 months:

  44. References: • Mesaros N, Nordmann P, Plesiat P, Roussel - Delvallez M, Van Eldere J et al. Pseudomonas aeruginosa: Resistance and therapeutic options at the turn of the new millennium. Clin. Microbiol. Infec. 2007; 13: 560-578. • Tomas M, Doumith M, Warner M, Turton JF, Beceiro A et al. Efflux pumps, OprD Porin, Amp C β-lactamase and multi resistance in Pseudomonas aeruginosa isolates from cystic fibrosis. Antimicrob. Agents Chemother 2010; 54:2219-2224. • Deziel E, Comeau Y, Villemur R. Initiation of Biofilm formation by P.aeruginosa 57RP correlates with emergence of hyper-piliated and highly adherent phenotypic variants deficient in swimming and twitching motilities. J. Bacteriol. 2001; 183:1195-1204. • Farrag HA. Post irradiation effect of adherent growth slime formation and antibiotic resistance of Pseudomonas aeruginosa causing human infection. J. Med. Sci. 2001;1:244-250 • Aendekerk S, Diggle SP, Song Z, Hoiby N, Cornelis P, Williams P and Camara M. The MexGHI- OpmD multidrug efflux pump controls growth, antibiotic susceptibility and virulence in Pseudomonas aeruginosa via 4-quinolone dependent cell to cell communication. Microbiology 2005; 151:1113-1125. • Deptula A and Gospodarek E. Reduced expression of virulence factors in multidrug resistant Pseudomonas aeruginosa strains. Arch. Microbiol 2010; 192:79-84. • Livermore DM and Woodford N. The β-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter. Trends Microbiol. 2006; 14:413-420. • Pitout JD, Gregson DB, Poirel L, McClure JA, Le P, Church DL. Detection of Pseudomonas aeruginosa producing metallo-β-lactamases in a large centralized laboratory. J Clin Microbiol 2005; 43: 3129-35. • Pellegrino, F.L.; Teixeira, L.M.; Carvalho Md, M.G.; Aranha, N.S., Pinto De, O.M., Mello Sampaio, J.L., D'Avila, F.A., Ferreira, A.L., Morim Ed, E.L., Riley, L.W., Moreira, B.M. Occurrence of a multidrug resistant Pseudomonas aeruginosa clone in different hospitals in Rio de Janeiro, Brazil. J ClinMicrobiol. 2002;40: 2420-2424.

  45. Sahly H, Navon-Venezia S, Roesler L, Hay A, Carmeli Y, Podschun R, Hennequin C, Forestier C, Ofek I. Extended spectrum β-Lactamase Production Is associated with an increase in cell invasion and expression of fimbrial adhesins in Klebsiella pneumoniae. Antimicrob. Agents Chemother.2008; 52:3029-3034. • Parveen M.; Harish B. N.; Parija, S. C. AmpC beta lactamases among gram negative clinical isolates from a tertiary hospital, South India. Braz J Microbiol. 2010; 41: 596-602. • Umadevi S, Joseph NM , Kumari K, Easow JM, Kumar S, Stephen S, Srirangaraj S, Raj S. Detection of extended spectrum beta lactamases, Amp C beta lactamases and Metallo-beta-lactamases in clinical isolates of ceftazidime resistant P. aeruginosa. Braz J Microbiol 2011; 42: 1284-1288. • De AS, Kumar SH, Baveja SM. Prevalence of metallo-β-lactamase producing P.aeruginosa and Acinetobacter species in intensive care areas in a tertiary care hospital. Indian J Crit Care Med 2010; 14:217-9. • Woods DE, Lam JS, Paranchych W, Speert DP, Campbell M, Godfrey AJ. Correlation of P.aeruginosavirulence factors from clinical and environmental isolates with pathogenicity in the neutropenic mouse. Can J Microbiol1997; 43: 541-51. • Davis KA, Santaniello JM, He LK, Muthu K, Sen S, Jones SB, et al. Burn injury and pulmonary sepsis: development of a clinically relevant model. J Trauma2004; 56: 272-8 • Collee JG and Marr W. Mackie and McCartney’s Practical Medical Microbiology; 14th, edition 1996. Chapter 5. Specimen collection, culture containers and media • Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC Jr. The fermentative gram negative bacilli. In: color atlas and textbook of diagnostic microbiology, Philadelphia Lippincott 6thed 2006:303-91 • Performance standards for antimicrobial susceptibility testing: nineteenth informational supplement. Clinical and laboratory standards institute. 2010; 29(3): M100-S19. • Arora S and Bal M. Amp C β-lactamase producing bacterial isolates from Kolkata hospital. Indian J Med Res 2005; 122:224-233.

  46. Coudron PE. Inhibitor based methods for detection of plasmid mediated AmpC β-lactamases in Klebsiellaspp., Escherichia coli and Proteus mirabilis. J ClinMicrobiol 2005; 43: 4163-7. • Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imepenem–EDTA disc method for differentiation of Metallo beta lactamase producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J ClinMicrobiol 2002; 40:3798-801. • Manoharan A, Chatterjee S, Mathai D, SARI Study Group. Detection and characterization of metallo beta lactamases producing Pseudomonas aeruginosa. Indian J Med Microbiol 2010; 28:241-4. • Podschun R., Sievers D, Fischer A, Ullmann U. Serotypes, hemagglutinins, siderophore synthesis, and serum resistance of Klebsiella isolates causing human urinary tract infections. J. Infect. Dis. 1993;168:1415-1421 • Raksha R, Srinivasa H, Macaden RS. Occurrence and characterization of uropathogenic Escherichia coli in urinary tract infections. Indian J Med Microbiol 2003; 21:102-7. • Siegfried L, Marta Kmetova, HanaPuzova, et al. Virulence associated factors in Escherichia coli strains isolated from children with urinary tract infections. J Med Microbiol 1994; 41:127-152.  • Stehling EG, Silveira WD Da, Silva D da. Study of Biological Characteristics of Pseudomonas aeruginosaStrains Isolated from Patients with Cystic Fibrosis and from Patients with Extra-Pulmonary Infections. Braz J Infect Dis 2008; 12(1):86-88. • Mittal R, Khandwaha RK, Gupta V, Mittal PK, Harjai K. Phenotypic characters of urinary isolates of Pseudomonas aeruginosa and their association with mouse renal colonization. Indian J Med Res 2006; 123:67-72. • Rashid MH, Kornberg A: Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa.Proc NatlAcadSci USA 2000, 97:4885-4890. • Prasad SV, Ballal M, Shivananda PG. Slime production a virulence marker in P.aeruginosa strains isolated from clinical and environmental specimens. A comparative study of two methods. Indian J Path Microbiol 2009; 52(2):191-193

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