This document summarizes information about vancomycin resistance in staphylococci. It discusses the definition of vancomycin resistance based on MIC levels, mechanisms of resistance such as cell wall alterations, risk factors for resistance like glycopeptide exposure, and recommendations for screening and treating resistant strains. Several studies reporting rates of vancomycin resistance in coagulase-negative staphylococci and rare cases of vancomycin resistance in Staphylococcus aureus are also summarized.

1. Vancomycin Resistance In
Staphylococci
Dr. Kanwal Deep Singh Lyall
M.D. Microbiology

2. Introduction
• Glycopeptide
• 1958
• NNIS 2000 data- 47% MRSA, 75%MRCONS in
ICUs
• DOC – MRSA , MRCONS
• First report –VR CONS -1979,1983
• First report - ↓susceptibility to SA in 1997

3. Definition
• NCCLS guidelines for Vancomycin
• For Staph
MIC(µg/ml)
• Susceptible ≤ 4
• Intermediate 8-16
• Resistant ≥ 32

4. • For Teicoplanin
• MIC
• Susceptible ≤ 8 µg/ml
• Intermediate 16
• Resistant ≥ 32

5. Heteroresistance
• Both in CoNS and SA
• Variability of Vancomycin susceptibilities among
subpopulations of single isolate
• two populations of cells
• Majority Vanco susceptible
• Minority Vanco resistant
• more common than pure resistance
• In Japan – 20% SA isolates
• In US- 0.3%

6. Laboratory Detection
• MIC by broth or agar dilution
• Etest
• Agar based methods -advantages
• more sensitive
Detect resistant subpopulations
• Single colonies visualized

7. • NCCLS – inoculum density of 5 x 105 CFU/ml
• Growth conditions- ↑Nacl
- ↓temp
• Inducing agent- Aztreonam

8. CDC Recommendations
• For S. aureus
(i) primary testing of S. aureus requires at least 24 h
of incubation,
(ii) susceptibility determination with disk diffusion
is not an acceptable method,
(iii) an MIC testing method should be used to confirm
vancomycin susceptibility
• Any S. aureus isolate for which the MIC is 4 µg/ml
should be sent to the CDC for confirmatory testing.

9. Infection Control Guidelines
• Precaution MCV
• Private room Yes Yes
• Gloves Yes Yes
• Gowns to enter room Yes No
• Gowns for patient contact Yes Yes
• Antibacterial hand washing agent Yes Yes
• Record of all health care workers entering room Yes No
• Mask and/or eye protection from aerosol Yes Yes
• Mupirocin for nasal colonization Yes No
• Limit number of health care workers caring for patient Yes Yes
• Nares cultures for all health care workers who entered room Yes Yes
• Health care workers at high risk for staphylococcal colonization should Yes No
not care for patient
HICPAC

10. • Isolation for duration of hospital stay Yes No
• Environmental cultures after terminal room cleaning Yes Optional
• Isolation on readmission until nares and previously infected, open sites Yes No
are staphylococcus negative
• Close unit to new admissions if nosocomial transmission occurs Yes No
• All specimens carried to laboratory (no Yes No
pneumatic tubes)
• When possible, postpone tests that Yes No
require patient to leave room
• Post staff at doorway to ensure Yes No
compliance with precautions

11. VRCoNS
• First report of clinically significant isolate -1987
• Summary of in vitro studies that examined reduced susceptibility to vancomycin among
coagulase-negative staphylococci
• Reference Location No. of isolates % of resistant isolates Susceptibility testing
• Henwood et al England 769 0.5 E test
• Santos Sanches et al Worldwide 1,351 0.07 Agar dilution
• Luh et al Taiwan 405 5.0 Agar dilution
• de Neeling et al Netherlands 7,334 0.4 Agar dilution
• Felmingham et al Europe 1,444 0.2 Agar dilution
• Gruneberg et al Europe 1,480 0.03 Agar dilution
• Herwaldt et al Iowa 28 0.04 Agar and broth dilution
• Goldstein et al France 362 0.03 Agar dilution
• Froggatt et al Virginia 70 42 Agar dilution

12. Risk Factors
• Peritoneal Dialysis
• Glycopeptide exposure – vancomycin
Teicoplanin

13. Species Variation
• Paradisi et al- all S.epidermidis isolates
sensitive
• Luh et al – S. epidermidis ↓susceptibility
• More common in S haemolyticus
• Also in S simulans in one study

14. Mechanism of resistance
• Vancomycin - binds irreversibly to the
terminal
D- alanyl –D alanine of cell wall
• Inhibits bacterial cell wall synthesis
• In VRCONS- altered cross links inhibit binding
to peptides
• Exact mechanism
not known

15. Treatment options
• Schwalbe et al – rifampin to vanco
• Aubert et al – rifampin and fusidic acid
• Sanyal et al – erythromycin
• Others- quinipristin-dalfopristin
- linezolid

16. VRSA
• 1997
• Less common than VRE and VRCoNS
• To date- no verified isolates of S.aureus with
true resistance to Vanco
• VISA more common
• First reported in 1995 in french child receiving
vanco for MRSA infection

17. Risk Factors
• Exposure to Glycopeptides
• MRSA infection
• Renal Failure

18. • VISA cases in the United States
• State ,yr Source Underlying illness Vancomycin
• Michigan, 1997 Peritoneal fluid Renal failure, 18
• MRSA peritonitis, cancer
• New Jersey, 1997 Blood ARF, MRSA bacteremia
18
• New York, 1998 Blood Renal failure, MRSA bacteremia 6
• Illinois, 1999 Blood Renal failure, MRSA endocarditis 3.5
• Minnesota, 2000 Blood Renal failure, MRSA osteomyelitis 18
• Nevada, 2000 Abscess fluid Complicated cholecystectomy with
10
polymicrobial
intrahepatic abscess (including MRSA)
• Maryland, 2000 Blood MRSA endocarditis

19. Mechanism of resistance
• True mechanism not known
• Emission of sex pheromone by S. aureus that
are in proximity to VRE that contain plasmids
encoding van genes could result in transfer of
these genes (Showsh et al)
• Van genes not recovered till date

20. • Other-
• Thicker and disorganized cell wall(Hanaki et
al)
• Less cross linking in peptidoglycans(Sieradzki K
et al)
• Thick cell wall trap vanco at periphery
• Altered cell wall - ↓affinity
• Role of PBPs unclear

21. • VRSA and VISA – colonies smaller
• Require more incubation time for coagulase –
more than 4 hours
• Misclassified as CONS

22. Treatment options
• TMP-SMX
• Tetracycline
• Quinipristin –Dalfopristin
• Linezolid

23. NCCLS Recommendations for VRSA
MIC (µg/ml)
Susceptible ≤ 4
Intermediate 8-16
Resistant ≥ 32

24. BSAC Recommendations for VRSA
MIC (µg/ml)
Susceptible ≤ 4
Resistant ≥ 32

25. CLSI
Int J Antimicrob Agents. 2007 Nov ;30(5):398-408
MIC (µg/ml)
• VISA 4-8
• VRSA ≥ 16

26. • Broth microdilution vancomycin MIC of 8-
16μg/ml
• E test vancomycin MIC of >6μg/ml
• growth on BHI agar containing 6μg/ml
vancomycin within 24 hours.
CDC criteria to identify VISA

27. • NCCLS disk-diffusion method and the Stokes
method are not accurate predictors of
reduced vancomycin susceptibility in
staphylococci

28. Screening of VRSA strains
Indian Journal of Medical Microbiology, (2005) 23 (1):52-55
• Hiramatshu method:
• Overnight grown cultures adjusted to 0.5
McFarland
• 10 μl spot inoculated on BHI agar (4 μg/ml)
of vancomycin
• incubated at 35°C for 24-48 hours

29. • CDC method:
• Overnight grown cultures adjusted to 0.5
McFarland
• diluted 100 times
• Spots 10 μl of the cultures inoculated on BHI
agar (6 μg/ml) of vancomycin.
• Incubated at 35°C for 24-48 hours.

30. • Method by Tenover et al:
• Overnight grown cultures adjusted to 0.5
McFarland
• diluted 100 times
• Spot 10μL of both the inoculums
• inoculated on MHA agar containing 5μg/ml
of vancomycin.
• Incubated at 35°C for 24-48 hours.

31. • Sensitivity Specificity
• CDC method 1 1
• MHA method 1 0.98
• Hiramatshu 1 0.85

32. Heteroresistant VRSA
• produce colonies on vancomycin-containing
BHI agar with vancomycin MICs of < 4 µg/ml
• Such strains were first reported from Japan in
1996

33. • Hanaki et al.
• hetero-VRSA -
• 3 to 5 fold greater quantities of PBP 2 and 2‘
• and ↑ quantities of cell-wall precursors, trap
vancomycin extracellularly . J Antimicrob Chemother
1998;42:199-209.
• amidation of glutamine residues in cell-wall
muropeptides , reduces the cross-linking within
the cell walls, reducing the number of
intracellular vancomycin target molecules J
Antimicrob Chemother. 1998 Sep;42(3):315–320.

34. Emergence of vancomycin resistant Staphylococcus aureus (VRSA) from a tertiary care hospital
from northern part of India
H K Tiwari, M R Sen
BMC Infect Dis. 2006; 6: 156
• 783 S aureus 93 CoNS
↓
• MIC testing ( vanco,teico, oxacillin)
• Brain Heart Infusion (BHI) vancomycin screen
agar test
• Disc diffusion testing
• PCR for mecA, vanA and vanB genes

35. • Results
• 2 - S. aureus - Vanco and teico resistant
• 6 - S. aureus – Vanco intermediate
• 2 - S. aureus – teico intermediate
• 1 – CoNS – Vanco and teico resistant
• 2 - CoNS strains –Vanco and teico
intermediate

36. • All VRSA, VISA and VRCoNS had shown growth
on BHI vancomycin screen agar (vancomycin 6
μg/ml) and were mecA PCR positive.
• None of these isolates have demonstrated
vanA/vanB gene by PCR.

37. Emergence of vancomycin resistance in Staphylococcus aureus. Glycopeptide-Intermediate
Staphylococcus aureus Working Group
Smith TL et al
N Engl J Med. 1999 Feb 18;340(7):493-501
• 2 patients with GISA (MIC Vanco 8-16µg/ml)
• 59 yr-old man DM and CRF.
• 18 weeks of vancomycin treatment for
recurrent MRSA peritonitis associated with
dialysis.
• Peritonitis due to GISA
• Removal of peritoneal catheter plus treatment
with rifampin and TMP-SMX eradicated the
infection.

38. • 66 yr old man with DM
• 18 weeks of vancomycin for recurrent MRSA
bacteremia.
• Bloodstream infection due to GISA
• This infection was eradicated with
vancomycin, gentamicin, and rifampin

39. • On electron microscopy, the isolates from the
infected patients had thicker extracellular
matrixes than control MRSA strains.