1

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

Quaid-i-Azam University, IslamabadOBAID ULLAH

Member , American Society for Microbiology (ASM), USA.

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

3

IntroductionIntroduction - - EnterococciEnterococci

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.

4

Resistance potential of Resistance potential of EnterococciEnterococci 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

5

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

Enterococcal Infections and Enterococcal Infections and Risk FactorsRisk Factors

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

7

Epidemiology in VREEpidemiology 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.

8

Treatment of VRETreatment 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

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.

10

Experimental Work

11

MATERIAL

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

12

SamplingSampling

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).

13

Isolation of Enterococci

Culturing on the Chromocult® Enterococci Agar (Merck).

Evaluation Red colonies with a diameter of 0.5 to 2 mm =

Enterococci

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

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

16

Antibiotic Susceptibility Testing

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

method

17

Antibiotics used for disk diffusion test

Antibiotic Abbreviation Potency Manufacturer Antibiotic class

Ampicillin AMP 25Oxoid Penicillin

Cefotaxime CTX 30Oxoid Cephem

Cefpirome CPO 30Oxoid Cephem

Chloramphenicol C 30Oxoid Phenicol

Ciprofloxacin CIP 5Oxoid Fluoroquinolone

Clindamycin DA 2Oxoid Lincosamide

Doxycycline DO 30Oxoid Tetracycline

Erythromycin E 15Oxoid Macrolide

Gentamicin CN 10Oxoid Aminoglycoside

Levofloxacin LEV 5Oxoid Fluoroquinolone

Linezolid LZD 30Oxoid Oxazolidinone

Sulbactum/cefoperazone SCF 105Oxoid β-lactamase

inhibitor/Cephem

Teicoplanin TEC 30Oxoid Glycopeptide

18

MINIMUM INHIBITORY CONCENTRATION MINIMUM INHIBITORY CONCENTRATION (MIC)(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

19

Antibiotic powders used for Antibiotic powders used for

determination of MICdetermination 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 biomedicals95%

EthanolH2O

5 Vancomycin 1000µg/mg MP biomedicals H2O H2O

20

RESULTSRESULTS

21

Colonies of Enterococci on

Chromocult® Enterococci agar.

Distribution of Enterococci isolated from different hospitals.

Identification of Enterococci

22

Distribution of Enerococci in different sample sources of hospitals

23

Biochemical identification of Biochemical identification of speciesspecies

Tubes showing the result of Sugar fermentation by Enterococci

24Distribution 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

26

Frequency of Vancomycin resistant Enterococci VRE) in three hospitals

27

Antibiotic Resistance profile of 54 VRE strains

28

Antibiotic sensitivity test plateAntibiotic sensitivity test plate

29

MIC Values of Cefotaxime and Erythromycin against 54 VRE strains

52

6

41

0

5

10

15

20

25

30

35

40

45

64 mg/L 128 mg/L 256 mg/L ≥512mg/L

MIC, Cefotaxime

13

52 1 1

41

0

5

10

15

20

25

30

35

40

45

0.5mg/L

2 mg/L

4 mg/L

8mg/L

64 mg/L

128 mg/L

≥512mg/L

MIC, Erythromycin

MIC Values

MIC Values

No.

of

Isola

tes

No.

of

Isola

tes

30

MIC Values of Ciprofloxacin and Doxycycline against 54 VRE strains

1

10

7

2

7

20

7

0

5

10

15

20

25

4 mg/L

8 mg/L

16 mg/L

32 mg/L

64 mg/L

128 mg/L

≥256 mg/L

MIC, Ciprofloxacin

3

1110

15

11

4

0

2

4

6

8

10

12

14

16

4 mg/L 8 mg/L 16 mg/L

32 mg/L

64 mg/L

128 mg/L

MIC, Doxycycline

MIC Values

MIC Values

No.

of

Isola

tes

No.

of

Isola

tes

31

MIC results of Vancomycin against VRE strains

52

2

0

10

20

30

40

50

60

04 mg/L 512 mg/L

MIC, Vancomycin

No.

of

Isola

tes

MIC Values

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.

33

RecommendationsRecommendations

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

34

Thanks for giving kind attention