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Enterococcus
 

Enterococcus

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

Enterococcus Enterococcus Presentation Transcript

  • 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).
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • Experimental Work 10
  •  MATERIAL  Blood agar (Oxoid),  Chromocult Enterococci Agar (Merck),  ChromID® VRE (Biomerieux),  Mueller Hinton agar (Oxoid),  Antibiotic discs (Oxoid),  Antibiotic powders (MP biomedics). 11
  • 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
  • Isolation of Enterococci  Culturing on the Chromocult® Enterococci Agar (Merck).  Evaluation Red colonies with a diameter of 0.5 to 2 mm = Enterococci 13
  • 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
  • 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
  • Antibiotic Susceptibility Testing 13 antibiotic discs were tested against VRE isolates Performed on Mueller Hinton agar by Kirby-Bauer disc diffusion method 16
  • 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
  • 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
  • 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
  • RESULTS 20
  • Identification of Enterococci Colonies of Enterococci onChromocult® Enterococci agar. 21 Distribution of Enterococci isolated from different hospitals.
  • Distribution of Enerococci in different samplesources of hospitals 22
  • Biochemical identification of species Tubes showing the result of Sugar fermentation by Enterococci 23
  • 24Distribution of Enterococci Species in different hospitals.
  • 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
  • Frequency of Vancomycin resistant Enterococci VRE)in three hospitals 26
  • Antibiotic Resistance profile of 54 VRE strains 27
  • Antibiotic sensitivity test plate 28
  • 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
  • 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
  • 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
  • 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
  • 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
  • Thanks for giving kind attention 34