Oritavancin Disk Diffusion can Distinguish Vancomycin-Intermediate
Staphylococcus aureus from Vancomycin-Susceptible S. aureus
Adam Belley, Francis Arhin and Greg Moeck
Contact:
Adam Belley
The Medicines Company
7170 Frederick Banting, 2nd Floor
St. Laurent, QC H4S 2A1
[email protected]
The Medicines Company, St-Laurent, QC, Canada
Revised Abstract
Introduction and Purpose
Results
Conclusions
Objectives: Vancomycin disk diffusion cannot distinguish
vancomycin intermediate Staphylococcus aureus (VISA;
broth microdilution MIC between 4-8 mg/l) from vancomycin
susceptible S. aureus (VSSA; broth microdilution MIC =< 2
mg/l) despite the differences in MIC of these phenotypes.
The lipoglycopeptide oritavancin is approved in the US for
acute bacterial skin and skin-structure infections caused by
susceptible isolates Gram-positive pathogens and is
currently under review by the European Medicines Agency.
We describe the use of oritavancin disks to distinguish VISA
from VSSA based on smaller inhibition zone diameters for
VISA.
A single 1200 mg dose of oritavancin is approved in the
US to treat adult patients with acute bacterial skin and skin
structure infections caused by susceptible isolates of
designated Gram-positive organisms1. In January 2015,
the Committee for Medicinal Products for Human Use
(CHMP) recommend the granting of a marketing
authorisation for oritavancin for the treatment of ABSSSI in
adults.
Table 1. Summary of MIC determinations and disk diffusion results using commercially-manufactured prototype oritavancin disks against seven clinical isolates of VSSA and
fourteen VISA isolates.
• The fourteen VISA isolates exhibit elevated
oritavancin MICs (1-4 mg/l) expected for VISA9
and are in the non-susceptible range to
oritavancin (MIC> 0.12 mg/l) according to US FDA
breakpoints1.
Methods: Fourteen VISA isolates obtained from the Network
of Antimicrobial Resistance in S. aureus and 7 clinical
isolates of VSSA with oritavancin MICs within the wild-type
distribution were used in this study. The isolates ATCC
29213 and ATCC 25923 were used for quality control (QC)
performance of broth microdilution MIC testing and disk
diffusion, respectively. For each isolate, MIC and inhibition
zone diameters where determined in parallel using the same
inoculum preparation. All testing followed CLSI guidelines
M07-A9 and M02-A11. Vancomycin (30 micrograms) disks
were from a commercial source. Prototype oritavancin disks
from a commercial manufacturer were formulated with 5
micrograms of oritavancin and a mixture of the excipients
polysorbate-80 and Span-80.
Results: Oritavancin and vancomycin MICs for ATCC 29213
and vancomycin inhibition zone diameters for ATCC 25923
were within the CLSI ranges. The oritavancin MICs against
the 7 VSSA and 14 VISA isolates ranged from 0.03 to 0.12
mg/l and 1 to 4 mg/l, respectively (table). Oritavancin
inhibition zone diameters for the 7 VSSA isolates ranged
from 21 to 25 mm and were at least 15 mm larger than the
range observed against the VISA isolates. In contrast,
vancomycin inhibition zone diameter ranges for the VSSA
and VISA isolates were overlapping and could not
discriminate the two phenotypes despite differences in
vancomycin MIC (table).
Conclusion: Using oritavancin disks, VISA isolates
exhibited at least 15 mm-smaller inhibition zone diameters
compared to VSSA isolates and therefore the oritavancin
disk diffusion assay may offer a screening method to
distinguish VISA isolates. Testing of additional VISA isolates
with oritavancin disk diffusion is warranted.
Phenotype
(number of
isolates)
MIC range (mg/l)
Inhibition zone
diameter range (mm)
Oritavancin
Vancomycin
Oritavancin
disk
Vancomycin
disk
VSSA (7)
0.03-0.12
0.5-1
21-25
16-18
VISA (14)
1-4
4-8
6
16-23
Currently, only the broth microdilution MIC method is
accepted for oritavancin antimicrobial susceptibility
testing2,3. Oritavancin poor diffusion in agar has hindered
development of a disk diffusion assay4. The recent
discovery of a blend of excipients (polysorbate-80 and
span-80) that promote diffusion of oritavancin in agar has
renewed interest in developing a disk diffusion assay for
oritavancin antimicrobial susceptibility testing5,6.
Isolates of vancomycin-intermediate S. aureus (VISA),
with vancomycin MICs that range from 4 to 8 mg/l, have
been associated with therapeutic failure7. EUCAST and
CLSI have indicated that vancomycin disk diffusion cannot
be used for antimicrobial susceptibility testing because
vancomycin disks cannot differentiate VSSA (vancomycin
MIC≤ 2 mg/l) from VISA isolates3,8.
The purpose of this study was to test whether disk
diffusion using oritavancin disks formulated with the
excipients polysorbate-80 and span-80 can distinguish the
fourteen tested VISA isolates (with oritavancin MICs
ranging from 1 to 4 mg/l) from seven random VSSA clinical
isolates (with oritavancin MICs ranging from 0.03-0.12
mg/l) based on inhibition zone diameters.
Phenotype
VSSA isolate
Clinical isolate
VISA isolate
1
2
3
4
5
6
7
NRS1
NRS3
NRS12
NRS14
NRS22 NRS24 NRS26 NRS29 NRS37 NRS56 NRS74 NRS79 NRS118 NRS402
Oritavancin MIC (mg/l)
0.06
0.12
0.03
0.06
0.06
0.03
0.12
2
2
2
4
2
1
1
2
1
1
1
1
2
2
Oritavancin inhibition
zone diameter (mm)
23
24
25
21
24
25
22
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Vancomycin MIC (mg/l)
1
1
1
1
1
0.5
1
8
8
8
8
4
4
8
4
4
8
8
4
8
8
Vancomycin inhibition
zone diameter (mm)
16
17
18
16
17
18
17
18
17
16
16
19
17
23
18
17
19
18
18
19
18
Figure 1. Inhibition zone diameters obtained with commercially-manufactured prototype oritavancin disks (ORI 5; left side of plate) or vancomycin disks (VA 30; right side of plate)
against seven clinical isolates (CI) of VSSA and fourteen VISA isolates.
1
2
3
4
5
6
VSSA
1. Orbactiv Prescribing Information,
http://www.themedicinescompany.com/app/webroot/img/orbact
iv-prescribing-information.pdf
2. CLSI M7-A10. Methods for dilution antimicrobial susceptibility
tests for bacteria that grow aerobically; approved standardtenth edition.
3. CLSI M100-S25. Performance standards for antimicrobial
susceptibility testing; twenty-fifth informational supplement.
January 2015.
NRS1
NRS3
NRS12
NRS14
NRS22
NRS24
NRS26
Fourteen VISA isolates obtained from the Network of
Antimicrobial Resistance in S. aureus and 7 random clinical
isolates of VSSA (obtained from Eurofins Medinet) with
oritavancin MICs within the wild-type distribution (MIC ≤0.12
mg/l) were used in this study. The S. aureus isolates ATCC
29213 and ATCC 25923 were used for quality control (QC)
performance of broth microdilution MIC testing and
vancomycin disk diffusion, respectively.
Presented at ECCMID 2015 (25th European Congress of Clinical Microbiology and Infectious Diseases) 25-28 April 2015, Copenhagen, Denmark
4. Blosser R, Karlowsky J, Loutit J, Porter S, Jones M,
Thornsberry C, Sahm, D. 2003. Evaluation of agar-based
susceptibility testing of oritavancin against gram-positive cocci.
103rd ASM abstract C-70.
5. Belley A, Arhin F, Sarmiento I, Moeck G. Development of a
disk diffusion assay for oritavancin. 2012; 52nd ICAAC abstract
D-762.
6. Koeth L, DiFranco-Fisher J, Arhin, F, Marquis M, Moeck G,
Belley A. Development of disk diffusion method for oritavancin:
comparison of disk diffusion with broth microdilution reference
method for Staphylococcus aureus. 2013; 23rd ECCMID
abstract P-1605.
7. Hiramatsu K, Hanaki H, Ino T, et al. Methicillin-resistant
Staphylococcus aureus clinical strain with reduced
vancomycin susceptibility. Journal of Antimicrobial
Chemotherapy 1997; 40:135-136.
Oritavancin disks
For each isolate, MIC and inhibition zone diameters where
determined in parallel using the same inoculum preparation
(using a picked-colony suspension) following CLSI
guidelines M07-A9 and M02-A11, respectively.
Commercially-prepared Mueller-Hinton agar (Oxoid
Microbiology Products, Nepean, ON, Canada) was used.
Vancomycin (30 micrograms) disks were from a commercial
source (BD, Sparks, MD, USA). All agar plates were
Incubated at 36oC for 24 hours prior to measurement of the
inhibition zone diameters. No inhibition zone diameter is
indicated as 6 mm (the diameter of the disk).
• Testing of additional VSSA and VISA isolates with
oritavancin disk diffusion is warranted.
References
Bacterial Isolates
MIC and disk diffusion determinations
• The oritavancin disk diffusion assay may offer a
screening method to distinguish VISA from VSSA.
7
Methods
Prototype oritavancin disks formulated with 5 micrograms of
oritavancin and a mixture of polysorbate-80 and Span-80
were obtained from a commercial manufacturer.
• Oritavancin disks reliably discriminated VISA from
VSSA since oritavancin disk inhibition zone
diameters were not observed for VISA isolates
and ranged from 21-25 mm against VSSA.
Oritavancin zone diameters therefore correlated
inversely with reference broth microdilution MICs
for isolates with these two phenotypes.
VISA
NRS29
NRS37
NRS56
NRS74
NRS79
NRS118
NRS402
8. EUCAST. Breakpoint tables for interpretation of MICs and
zone diameters. Version 5.0, 2015. http://www.eucast.org.
9. Vidaillac C, Parra-Ruiz J, Rybak M. In vitro time-kill analysis of
oritavancin against clinical isolates of methicillin-resistant
Staphylococcus aureus with reduced susceptibility to
daptomycin. Diagnostic Microbiology and Infectious Disease
2011; 71: 470-473.
Disclosures
Support, funding and medical writing assistance for this
study was provided by The Medicines Company.
Poster # 0713