Prevalence of Vancomycin-Resistant Enterococci (VRE) in the hospitalized patients of Islamabad and Rawappindi

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Prevalence of Vancomycin-Resistant Enterococci
(VRE) in the hospitalized patients of Islamabad
and Rawappindi
OBAID ULLAH

• Quaid-i-Azam University, Islamabad

Member , American Society for Microbiology (ASM),
USA. Associate Member, International Federation
of Infection Control (IFIC).
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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

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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.

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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

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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

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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

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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 AFIP

Uttley, 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 Infect
103173-181. Khan, E., Sarwari A., Hassan, R.,
Ghori, S., Babar, I., OBrien, F. and Grubb, W.
2002. Emergence of vancomycin resistant
Enterococcus faecium at a tertiary care hospital
in Karachi, Pakistan. J Hosp Infect 52 292-6.
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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

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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.

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Experimental Work
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• MATERIAL
• Blood agar (Oxoid),
• Chromocult Enterococci Agar (Merck),
• ChromID VRE (Biomerieux),
• Mueller Hinton agar (Oxoid),
• Antibiotic discs (Oxoid),
• Antibiotic powders (MP biomedics).

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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).

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Isolation of Enterococci

• Culturing on the Chromocult Enterococci Agar
  (Merck).
• Evaluation Red colonies with a diameter of 0.5
  to 2 mm Enterococci

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Identification of Enterococcus Species

• By the Biochemical tests
• Three tests were performed to identify the
  species
• Arabinose fermentation, Sorbitol fermentation
  and Growth at 4C

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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

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Antibiotic Susceptibility Testing

• 13 antibiotic discs were tested against VRE
  isolates
• Performed on Mueller Hinton agar by Kirby-Bauer
  disc diffusion method

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Antibiotics used for disk diffusion test
Antibiotic Abbreviation Potency Manufacturer Antibiotic class
Ampicillin AMP 25 Oxoid Penicillin
Cefotaxime CTX 30 Oxoid Cephem
Cefpirome CPO 30 Oxoid Cephem
Chloramphenicol C 30 Oxoid Phenicol
Ciprofloxacin CIP 5 Oxoid Fluoroquinolone
Clindamycin DA 2 Oxoid Lincosamide
Doxycycline DO 30 Oxoid Tetracycline
Erythromycin E 15 Oxoid Macrolide
Gentamicin CN 10 Oxoid Aminoglycoside
Levofloxacin LEV 5 Oxoid Fluoroquinolone
Linezolid LZD 30 Oxoid Oxazolidinone
Sulbactum/cefoperazone SCF 105 Oxoid ß-lactamase inhibitor/Cephem
Teicoplanin TEC 30 Oxoid Glycopeptide
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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

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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
4 Erythromycin 971µg/mg MP biomedicals 95 Ethanol H2O
5 Vancomycin 1000µg/mg MP biomedicals H2O H2O
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RESULTS
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Colonies of Enterococci onChromocult
Enterococci agar.
Identification of Enterococci
Distribution of Enterococci isolated from
different hospitals.
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Distribution of Enerococci in different sample
sources of hospitals
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Biochemical identification of species
Tubes showing the result of Sugar fermentation by
Enterococci
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Distribution of Enterococci Species in different
hospitals.
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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
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Frequency of Vancomycin resistant Enterococci
VRE) in three hospitals
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Antibiotic Resistance profile of 54 VRE strains
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Antibiotic sensitivity test plate
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MIC Values of Cefotaxime and Erythromycin against
54 VRE strains
No. of Isolates
No. of Isolates
MIC Values
MIC Values
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MIC Values of Ciprofloxacin and Doxycycline
against 54 VRE strains
No. of Isolates
No. of Isolates
MIC Values
MIC Values
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MIC results of Vancomycin against VRE strains
No. of Isolates
MIC Values
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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.

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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

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Thanks for giving kind attention