Enterococcus

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  • Obaid Khan 07/10/12
  • Obaid Khan 07/10/12
  • Enterococcus

    1. 1. Prevalence of Vancomycin-Resistant Enterococci (VRE) in the hospitalized patients of Islamabad and Rawappindi OBAID ULLAH Quaid-i-Azam University, IslamabadMember , American Society for Microbiology (ASM), USA. 1Associate Member, International Federation of Infection Control (IFIC).
    2. 2. Introduction - Nosocomial Infections Nosocomial infections pose a continuing challenge Defined as an infection which develops 48 hours after hospital admission or within 48 hours 1.7 million infections and 99,000 deaths annually Organisms of current concern  Methicillin-resistant Staphylococcus aureus,  Glycopeptide-intermediate and resistant S aureus,  Vancomycin-resistant enterococci, and  Multidrugresistant Gram-negative bacteria 2
    3. 3. Introduction - Enterococci The 3rd cause of nosocomial infections. Involved in over 800,000 infections per year in the USA in 2004 Gram(+) , Cocci. Survive in 6.5% NaCl and at a pH of 9.6 Most capable of growing from 10 º to 45 º C range; Survive at 60º C for 30 minutes There are 23 species of Enterococci. Two that account for the majority of human infections are: Enterococcus faecalis and Enterococcus faecium. Part of the normal bowel flora. 3
    4. 4. Resistance potential of Enterococci Innately resistant to most antibiotics including:  Cephalosporins, Penicillins, Clindamycin and Trimethoprim Can also acquire, accumulate and transfer genetic elements e.g. (plasmids, and transposons) using conjugation Acquire Resistance  Macrolides  Tetracycline  Lincosamides  Chloramphenicol  Aminoglycosides  Penicillin (without beta-lactamase)  Penicillin (with beta-lactamase)  Vancomycin  Quinolones 4
    5. 5. Enterococcal Infections and Risk Factors Wide range of infections  Endocarditis, Septicemia, Urinary Tract Infections, Intra-abdominal and Wound Infections as well as infections of Indwelling Lines. Having an underlying comorbid condition Prolonged length of hospital stay And close proximity to another VRE-colonized or -infected patient Vancomycin has been used as the last resort to treat enterococcal infections 5
    6. 6. Vancomycin Action and Resistance by Enterococci Binding to the terminal D-alanyl-D-alanine residues → prevents crosslinking of the peptidoglycan component in the cell wall of G(+) organisms Inhibits bacterial growth, eventually leading to death. D-alanyl-D-alanine residue ↓ D-alanyl-D-lactate moiety Vancomycin cannot bind to this peptide 6
    7. 7. Epidemiology in VRE  First described in Europe in 1989.  Primarily a nosocomial pathogen  Alarming increase  In the United States, prevalence as high as 47%  First case of VRE in Pakistan was reported in 2002 from Karachi  First case of VRE in Rawalpindi / Islamabad in 2003 by AFIPUttley, A.H., George, R.C., Naidoo, J., Woodford, N., Johnson, A.P., Collins, C.H., Morrison, D., Gilfillan, A.J., Fitch, L.E.and Heptonstall, J. 1989. High-level vancomycin-resistant enterococci causing hospital infections. Epidemiol Infect103:173−181.Khan, E., Sarwari A., Hassan, R., Ghori, S., Babar, I., O’Brien, F. and Grubb, W. 2002. Emergence of vancomycin resistantEnterococcus faecium at a tertiary care hospital in Karachi, Pakistan. J Hosp Infect; 52: 292-6. 7
    8. 8. Treatment of VRE Quinupristin-Dalfopristin (1999)  First antimicrobial agent available for the treatment  Inhibiting protein synthesis Linezolid (2000)  Inhibits ribosomal protein synthesis Daptomycin (2003)  Lipopeptide fermentation product of Streptomyces roseosporus  Disrupts multiple aspects of bacterial membrane Tigecycline (2005 )  A broad-spectrum glycylcycline antimicrobial agent Mannopeptimycins and Dalbavancin (Future treatments)  Semisynthetic glycopeptides 8
    9. 9. Aim and Objectives of Current Study To isolate and identify enterococci from different clinical specimens of three tertiary care hospitals of Rawalpindi and Islamabad. Detection of Vancomycin resistant enterococci from the isolated strains. Determination of frequency of VRE in Pakistan Institute of Medical Sciences, Shifa Internaional Hospital and Holy Family Hospital. Checking the antibiotic susceptibility of different antibiotics against Vancomycin resistant enterococci (VRE). To check the MIC (Minimum Inhibitory Concentration) of different antibiotics. 9
    10. 10. Experimental Work 10
    11. 11.  MATERIAL  Blood agar (Oxoid),  Chromocult Enterococci Agar (Merck),  ChromID® VRE (Biomerieux),  Mueller Hinton agar (Oxoid),  Antibiotic discs (Oxoid),  Antibiotic powders (MP biomedics). 11
    12. 12. Sampling Three different hospitals of Islamabad and Rawalpindi  Pakistan Institute of Medical Sciences (P.I.M.S), Islamabad.  Shifa International Hospital, Islamabad.  Holy Family Hospital, Rawalpindi. Specimens  Urine, Blood, Pus, Tissues, Surgical sites etc. A total of 133 samples were collected in a period of 6 months (April, 2009- September, 2009). 12
    13. 13. Isolation of Enterococci  Culturing on the Chromocult® Enterococci Agar (Merck).  Evaluation Red colonies with a diameter of 0.5 to 2 mm = Enterococci 13
    14. 14. Identification of Enterococcus Species By the Biochemical tests Three tests were performed to identify the species  Arabinose fermentation, Sorbitol fermentation and Growth at 4°C 14
    15. 15. Isolation of Vancomycin Resistant Enterococci  Enterococcus species were then sreaked on to the chromID™ VRE (Biomerieux) media  Contains two chromogenic substrates  alpha-Glucosidase & beta-Galactosidase  After 24hrs of incubation  Bluish-green colour = Vancomycin resistant E. faecalis  Violet colour = Vancomycin resistant E. faecium 15
    16. 16. Antibiotic Susceptibility Testing 13 antibiotic discs were tested against VRE isolates Performed on Mueller Hinton agar by Kirby-Bauer disc diffusion method 16
    17. 17. Antibiotics used for disk diffusion testAntibiotic Abbreviation Potency Manufacturer Antibiotic class Oxoid PenicillinAmpicillin AMP 25 Oxoid CephemCefotaxime CTX 30 Oxoid CephemCefpirome CPO 30 Oxoid PhenicolChloramphenicol C 30 Oxoid FluoroquinoloneCiprofloxacin CIP 5 Oxoid LincosamideClindamycin DA 2 Oxoid TetracyclineDoxycycline DO 30 Oxoid MacrolideErythromycin E 15 Oxoid AminoglycosideGentamicin CN 10 Oxoid FluoroquinoloneLevofloxacin LEV 5 Oxoid OxazolidinoneLinezolid LZD 30 Oxoid β-lactamaseSulbactum/cefoperazone SCF 105 inhibitor/Cephem Oxoid GlycopeptideTeicoplanin TEC 30 17
    18. 18. MINIMUM INHIBITORY CONCENTRATION (MIC)  MIC agaist Vancomycin Resistant Enterococci strains  Agar dilution method was used to determine the MICs  Stock solutions were prepared by using the formula 1000/P x V x C = W  P= potency given by the manufacturer (µg/mg),  V= volume required (ml),  C= final concentration of the solution (multiples of 1000) (mg/l),  W= weight of antibiotic in mg to be dissolved in volume V (ml).  These antibiotic stock solutions were used to make antibiotic dilutions  Antibiotic dilution range of 0.25, 0.5, 1.0, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024 μg/ml 18
    19. 19. Antibiotic powders used for determination of MIC S.No. Antibiotic Potency Source Solvent Diluent 1 Cefotaxime 950µg/mg MP biomedicals H2O H2O 2 Ciprofloxacin 995µg/mg MP biomedicals H2O H2O 3 Doxycycline 839µg/mg MP biomedicals H2O H2O 95% 4 Erythromycin 971µg/mg MP biomedicals H2O Ethanol 5 Vancomycin 1000µg/mg MP biomedicals H2O H2O 19
    20. 20. RESULTS 20
    21. 21. Identification of Enterococci Colonies of Enterococci onChromocult® Enterococci agar. 21 Distribution of Enterococci isolated from different hospitals.
    22. 22. Distribution of Enerococci in different samplesources of hospitals 22
    23. 23. Biochemical identification of species Tubes showing the result of Sugar fermentation by Enterococci 23
    24. 24. 24Distribution of Enterococci Species in different hospitals.
    25. 25. Frequency of Vancomycin Resistant Enterococci (VRE)Growth of vancomycin resistant enterococci on ChromID VRE media.Violet colonies on the media shows vancomycin resistant Eneterococci faecium 25
    26. 26. Frequency of Vancomycin resistant Enterococci VRE)in three hospitals 26
    27. 27. Antibiotic Resistance profile of 54 VRE strains 27
    28. 28. Antibiotic sensitivity test plate 28
    29. 29. MIC Values of Cefotaxime and Erythromycin against 54 VRE strains MIC, Cefotaxime 45 41 40 35No. of Isolates 30 25 45 41 MIC, Erythromycin 20 40 15 35 10 6 5 30 No. of Isolates 5 2 25 0 64 mg/L 128 mg/L 256 mg/L ≥512mg/L 20 MIC Values 15 10 5 5 3 2 1 1 1 0 0.5 2 4 8 64 128 ≥512 mg/L mg/L mg/L mg/L mg/L mg/L mg/L MIC Values 29
    30. 30. MIC Values of Ciprofloxacin and Doxycycline against 54 VRE strains 25 MIC, Ciprofloxacin 20 20 15No. of Isolates 10 10 7 7 7 5 16 15 2 MIC, Doxycycline 1 14 0 12 11 11 4 8 16 32 64 128 ≥256 10 mg/L mg/L mg/L mg/L mg/L mg/L mg/L 10 No. of Isolates MIC Values 8 6 4 4 3 2 0 4 mg/L 8 mg/L 16 32 64 128 mg/L mg/L mg/L mg/L MIC Values 30
    31. 31. MIC results of Vancomycin against VRE strains 60 52 50 MIC, Vancomycin 40 No. of Isolates 30 20 10 2 0 04 mg/L 512 mg/L MIC Values 31
    32. 32. Conclusions Most of the strains of the enterococci isolated were E. faecium followed by E. faecalis. Enterococci were mostly recovered by urine samples followed by pus, blood, wound and tissues. Enterococci displaying multidrug resistance and severe therapeutic problem, but their emergence in Pakistan still has not been well demonstrated Teicoplanin was the drug of choice against the enterococcal infections including those caused by VRE strains. Other than teicoplanin, linezolid and ampicillin could be used for treatment of enterococcal infections effectively. 32
    33. 33. Recommendations Prudent use of vancomycin Education of hospital staff regarding the problem Rapid and accurate identification of VRE in the microbiology laboratory Aggressive infection control measures utilizing contact isolation and cohorting where necessary to prevent person-to-person transmission Effective interaction between microbiology lab and hospitals 33
    34. 34. Thanks for giving kind attention 34

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