1. 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.
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Associate Member, International Federation of Infection Control (IFIC).

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
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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.
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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
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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
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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
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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 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
103:173−181.
Khan, E., Sarwari A., Hassan, R., Ghori, S., Babar, I., O’Brien, 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. 7

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
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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.
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10. Experimental Work
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11.  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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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).
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13. 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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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
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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
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16. 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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17. Antibiotics used for disk diffusion test
Antibiotic Abbreviation Potency Manufacturer Antibiotic class
Oxoid Penicillin
Ampicillin AMP 25
Oxoid Cephem
Cefotaxime CTX 30
Oxoid Cephem
Cefpirome CPO 30
Oxoid Phenicol
Chloramphenicol C 30
Oxoid Fluoroquinolone
Ciprofloxacin CIP 5
Oxoid Lincosamide
Clindamycin DA 2
Oxoid Tetracycline
Doxycycline DO 30
Oxoid Macrolide
Erythromycin E 15
Oxoid Aminoglycoside
Gentamicin CN 10
Oxoid Fluoroquinolone
Levofloxacin LEV 5
Oxoid Oxazolidinone
Linezolid LZD 30
Oxoid β-lactamase
Sulbactum/cefoperazone SCF 105
inhibitor/Cephem
Oxoid Glycopeptide
Teicoplanin TEC 30
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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
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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
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20. RESULTS
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21. Identification of Enterococci
Colonies of Enterococci on
Chromocult® Enterococci agar.
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Distribution of Enterococci isolated from different hospitals.

22. Distribution of Enerococci in different sample
sources of hospitals
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23. Biochemical identification of species
Tubes showing the result of Sugar fermentation by Enterococci
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24. 24
Distribution of Enterococci Species in different hospitals.

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
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26. Frequency of Vancomycin resistant Enterococci VRE)
in three hospitals
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27. Antibiotic Resistance profile of 54 VRE strains
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28. Antibiotic sensitivity test plate
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29. MIC Values of Cefotaxime and Erythromycin against 54 VRE
strains
MIC, Cefotaxime
45 41
40
35
No. 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
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30. MIC Values of Ciprofloxacin and Doxycycline against 54 VRE
strains
25
MIC, Ciprofloxacin
20
20
15
No. 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
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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
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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.
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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
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34. Thanks for giving kind attention
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