Layout

Comparison of Mupirocin 5 and 20 µg Disk Results to Microdilution and Etest MICs Lab Specialists, Inc. 1651 A Crossings Parkway, Westlake, against Susceptible Isolates and a Resistant Challenge Set of Staphylococcus aureus OH4 4145, USATel: +1 440 835 4458Fax: +1 440 835 5786 L. M. Koeth,1 J. Difranco,1 C. Jakielaszek,2 R. Shawar2 & L. Miller2
1Laboratory Specialists, Inc., Westlake, OH, USA; 2GlaxoSmithKline Pharmaceuticals, Collegeville, PA, USA
Figure 2. Correlation Between Zone Diameters Obtained by 20 µg Mupirocin Disk
Figure 4. Correlation Between Zone Diameters Obtained by 20 µg Mupirocin
(Two ISA Lots) and BSAC Disk Breakpoint Interpretive Criteria
Disk (One MHA Lot) and BSAC Disk Breakpoint Interpretive Criteria
Background: A 5 µg mupirocin disk currently available for testing has no definable zone category
Disk Diffusion using ISA (BSAC Inoculum)
for differentiating between low-level-resistant (LLR; MICs 8–256 µg/mL) and high-level-resistant 20
µg/mL) isolates. A 20 µg mupirocin disk has recently become commercially µg disk (Table 1, Figures 1 and 2)
available (Mast, Bootle, UK) for testing according to British Society for Antimicrobial Chemotherapy (BSAC) disk method, and has tentative interpretive criteria for LLR and HLR categories. The Lot #324391 = 78.9% agreement (21.1% were wrongly classified as LLR) purpose of this study was to assess the BSAC method by comparing zone diameters for 5 and Lot #335431 = 95% agreement (5% were wrongly classified as LLR).
20 µg disks with Clinical Laboratory Standards Institute (CLSI) microbroth and Etest MICs for Isolates (n) 10
60 Staphylococcus aureus in order to determine the performance of both disks in detecting LLR Lot #324391 = 96.2% agreement (3.8% were wrongly classified as HLR) Isolates (n) 10
and HLR isolates. BSAC and CLSI disk methods were also compared. Methods: Twenty-one
Lot #335431 = 96.2% agreement (3.8% were wrongly classified as HLR).
susceptible (MICs ≤4 µg/mL), 26 LLR (MICs 16, 32, 128 µg/mL) and 13 HLR S. aureus (MICs >512 µg/mL) were tested by BSAC disk method with a 5 and 20 µg mupirocin disk using two lots of Lot #324391 = 92.3% agreement (7.7% were wrongly classified as LLR) 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32>32 IsoSensitest agar (ISA) and for comparison purposes, one lot of Mueller Hinton agar (MHA) using Lot #335431 = 84.6% agreement (15.4% were wrongly classified as LLR).
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32>32 both CLSI and BSAC methods. The same isolates were also concurrently tested by microbroth Zone diameter (mm)
dilution (CLSI) and Etest. Results: 96.2% of LLR isolates were accurately categorized using the
5 µg disk (Table 1, Figure 3)
Zone diameter (mm)
20 µg disk. All HLR isolates were accurately categorized using the 5 µg disk. The 20 µg disk tested Figure 3. Correlation Between Zone Diameters Obtained by 5 µg Mupirocin
on two lots of ISA detected 84.6% and 92.3% of HLR isolates. The 5 µg disk tested on two lots of Lot #324391 = 94.7% agreement (5.3% were wrongly classified as resistant or LLR) Disk (Two ISA Lots) and BSAC Disk Breakpoint Interpretive Criteria
ISA detected 84.6% and 92.3% of LLR isolates. MHA zones were less defined and more difficult to Lot #335431 = 95% agreement (5% were wrongly classified as resistant or LLR).
Figure 5. Correlation Between Zone Diameters Obtained by 5 µg Mupirocin
read than ISA and were 2 mm lower by CLSI compared with BSAC. Of the 28 isolates with on-scale Disk (One MHA Lot) and BSAC Disk Breakpoint Interpretive Criteria
MICs, 100% of the microbroth dilution and Etest results were within ±1 doubling dilution; of these Lot #324391 = 84.6% (15.4% were wrongly classified as HLR) 20 (71.4%) were identical. Conclusions: There was good correlation of mupirocin Etest and
Lot #335431 = 92.3% (7.7% were wrongly classified as HLR).
microdilution MICs for all isolates tested. The use of the 20 µg mupirocin disk on ISA according to the BSAC procedure is an acceptable method for detection of mupirocin susceptible, LLR and Isolates (n)
Disk diffusion using BSAC inoculum on MHA or CLSI
Isolates (n) 30
Deviations from BSAC method, such as using MHA with BSAC recommended inoculum (Table 2) or
The current British Society for Antimicrobial Chemotherapy (BSAC) standardized disk susceptibility 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32>32 CLSI recommended inoculum (Table 3) with the 20
µg disk resulted in very high (76.2–90.5%) error µg and a 20 µg mupirocin disk for testing of Staphylococcus aureus. There rate in erroneously calling susceptible isolates as LLR. Similarly, deviations from BSAC method using is no intermediate zone range in the BSAC method for detection of LLR isotypes using the 5 µg disk.
Zone diameter (mm)
µg disk resulted in marked errors (33.3–71.4%) in calling susceptible isolates as resistant. When µg disk was recently added to the BSAC disk method and does provide an intermediate zone 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32>32 utilizing existing BSAC breakpoints, variations to BSAC method, including use of MHA and inoculum, range for detection of LLR isolates. The purpose of this study was to compare zone diameters for both will result in significant categorical errors (Table 2, Figures 4 and 5).
Table 2. Correlation of Mupirocin 5 and 20 µg disk (BSAC Inoculum on MHA) Results to
the 5 and 20 µg disks (using the BSAC methodology with IsoSensitest agar [ISA] and with Mueller Zone diameter (mm)
Hinton agar [MHA]) with Clinical Laboratory Standards Institute (CLSI) broth microdilution and Etest Susceptible, LLR and HLR Categorical Results as Determined by MIC (CLSI Method)
MIC results for a challenge set of organisms in order to determine the capability of the disk method to detect low- and high-level mupirocin resistance in isolates of S. aureus. BSAC and CLSI disk There was good correlation of mupirocin Etest and microdilution MICs. There were 28 isolates with methods, which use different culture media and inoculum preparations, were also compared.
MIC susceptibility category, n (%)
on-scale MICs (MICs that fall within the range of MIC values tested); 20 of these were identical and 20 µg disk (MHA Lot #5335405)
eight were half or one doubling dilution different. All other isolates had MICs less than or equal to mm Susceptible
the lowest concentration or greater than the highest concentration tested and were similar by both With both disks there were susceptible isolates categorized as LLR isolates. All susceptible isolates could be detected using the 20 or 5 µg mupirocin disk on ISA according to the BSACprocedure, if the susceptible breakpoint was moved to 24 mm for the 20 µg disk and 19 mm Microorganisms
Table 1. Correlation of Mupirocin 5 and 20 µg Disk (BSAC Method ISA)
Results to Susceptible, LLR and HLR Categorical Results as Determined
There were some HLR isolates that were categorized as LLR using the 20 µg disk; this did Sixty S. aureus isolates provided by IHMA (International Health Management Associates, Inc., by MIC (CLSI Method)
not occur with the 5 µg disk. The 5 µg disk may be considered slightly more effective than Schaumburg, IL, USA), selected according to mupirocin susceptibility; 21 susceptible (slight 5 µg disk (MHA Lot #5335405)
variation in total number of susceptible isolates with ISA was due to unreadable zones as a the 20 µg disk in detecting HLR isolates based on the breakpoints that were chosen for the mm Susceptible
Resistant
result of plate contamination), 26 LLR, and 13 HLR isolates.
Quality control strains: S. aureus ATCC 25923, S. aureus ATCC 29213, and S. aureus NCTC MIC susceptibility category, n (%)
20 µg disk (ISA Lot #324391 and #335431)
mm Susceptiblea
MHA – Becton Dickinson prepared plates, Sparks, MD, USA, Lot #5335405.
ISA – Oxoid, LSI prepared plates, Hampshire, UK, Lot #324391 and #335431. 5 µg disk (ISA Lot #324391 and #335431)
The use of the 20 µg mupirocin disk on ISA according to the BSAC procedure and mm Susceptiblea
Resistant
breakpoints is suitable for the detection of susceptible, low-level and high-level mupirocin- aBSAC recommended breakpoint; bAdjusted/suggested breakpoint Testing Methodology
HLR by MIC, LLR by disk; LLR by MIC, HLR by disk; Susceptible by MIC, LLR by disk Categorical errors will occur if modifications to the BSAC method (i.e. media and inoculum) Disk
are made and existing BSAC breakpoints are utilized.
BSAC inoculum (1:10 dilution of a 0.5 McFarland) Table 3. Correlation of Mupirocin 5 and 20 µg Disk (CLSI Inoculum on MHA) Results to
The use of the 20 µg disk on MHA using either BSAC or CLSI inoculum with adjusted BSAC inoculum (1:10 dilution of a 0.5 McFarland) Susceptible, LLR and HLR Categorical Results as Determined by MIC (CLSI Method)
breakpoints can reliably detect LLR and HLR S. aureus (see Tables 2 and 3).
aSlight variation in total number of isolates was due to plate contamination The use of the 5 µg disk on ISA according to the BSAC procedure was suitable for the HLR by MIC, LLR by disk; LLR by MIC, HLR by disk; Susceptible by MIC, LLR by disk detection of susceptible, resistant, and low- and high-level mupirocin-resistant S. aureus MIC susceptibility category, n (%)
when applying the suggested breakpoints. 20 µg (BSAC): HLR (resistant) ≤6 mm, LLR (intermediate) 7–26 mm, susceptible ≥27 mm 20 µg disk (MHA Lot #5335405)
5 µg (BSAC): resistant ≤21 mm, susceptible ≥22 mm.* The 5 µg mupirocin disk on MHA (using the BSAC or CLSI inoculum) did not differentiate Figure 1. Scatterplot of MIC by CLSI Broth Microdilution Method versus 20 µg Disk
mm Susceptible
*As there are no existing BSAC breakpoints for the 5 µg disk, following breakpoints were by BSAC Method (ISA; Lot #324391 and #335431)
assigned for the purpose of this study: HLR ≤6 mm, LLR 7–21 mm.
As this study included relatively small numbers of isolates and no isolates with mupirocin MICs of 8, 256 or 512 µg/mL, further investigation is warranted to confirm the suggested MIC
There was good correlation of Etest and broth microdilution MICs. 5 µg disk (MHA Lot #5335405)
BSAC MIC breakpoints: resistant >256, intermediate = 8–256; susceptible ≤4.1 mm Susceptible
Resistant
Acknowledgement
Antimicrobial Agents
This study was supported by GlaxoSmithKline Mupirocin powder (Lot #WRS46) – GlaxoSmithKline, Collegeville, PA, USA. A 5120 µg/mL stock solution was made using water as a dilutant and trays made according to CLSI guidelines.
Mupirocin 20 µg disks (Lot #190308) – Mast Group Ltd, Bootle, UK.
Andrews JM for BSAC Working Party. J Antimicrob Chemother 2005; 56: 60–76.
µg disks (Lot #3245025) – Becton Dickinson, Sparks, MD, USA. 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 >32 Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests, 9th edn. Document M2-A9, Vol 26, No. 1. Wayne, PA, USA: CLSI, 2006.
Data Analysis
Zone diameter (mm)
Clinical Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests BSAC recommended breakpoint; bAdjusted/suggested breakpoint Category agreement was calculated based on comparison of zone interpretation results to MIC HLR by MIC, LLR by disk; LLR by MIC, HLR by disk; Susceptible by MIC, LLR by disk for Bacteria that Grow Aerobically, 7th edn. Document M7-A7, Vol 26, No. 2. Wayne, PA, USA: The 46th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), 27–30 September 2006, San Francisco, CA, USA

Source: http://www.labspec.org/pdf/ICAAC%202006%20D-0809%20Comparison%20of%20Mupirocin%205%20and%2020%20disk%20results%20to%20microdilution%20and%20Etest%20MICs%20against%20susceptible%20isolates%20and%20a%20resistant%20challenge%20set%20of%20S.%20aureus.pdf