Title: The%20Use%20of%20Cefoxitin%20for%20the%20Determination%20of%20Methicillin%20Resistance%20in%20Staphylococci
1The Use of Cefoxitin for the Determination of
Methicillin Resistance in Staphylococci
- John D. Perry
- Microbiology Department
- Freeman Hospital
- Newcastle upon Tyne
2BSAC Working Party recommendations for detection
of methicillin resistance (2004)
- Inoculum Semi confluent
- Medium Columbia agar plus 2 salt.
- Incubation conditions 30C
- Discs Oxacillin 1 µg or methicillin 5 µg
3BSAC Working Party recommendations for detection
of methicillin resistance (2004)
- Disadvantages
- A separate medium (at a different temperature) is
required to that used for testing other
anti-staphylococcal agents. - False resistance may be encountered due to
hyper-production of ß-lactamase - False susceptibility may be encountered with
strains that are highly sensitive to salt.
4Felten et alEvaluation of three techniques
for detection of low-level methicillin-resistant
Staphylococcus aureus (MRSA) a disk diffusion
method with cefoxitin and moxalactam, the Vitek 2
system, and the MRSA-screen latex agglutination
test.J Clin Microbiol. 2002 Aug40(8)2766-71.
5- Felten et al. examined
- 69 mecA-negative S. aureus strains and
- 83 mecA positive S. aureus strains (MRSA)
including 69 with heterogeneous resistance. - Susceptibility to cefoxitin 30 µg discs was
determined on Mueller-Hinton agar at 37C using
high and low inocula. - Oxacillin disc testing was performed in parallel
using CLSI and CASFM methodologies.
6Felten et al. (continued)
- Results
- The cefoxitin 30 µg disc test showed 100
specificity and 100 sensitivity for detection
of MRSA. Interpretive criteria were - Zone diameter lt 27 mm MRSA.
- Disc testing using oxacillin showed a sensitivity
of 95.2 96.4 depending on the inoculum used.
Specificity was 100 .
7Significance of the study by Felten et al.
- The results suggested that cefoxitin was
potentially more sensitive that oxacillin for
detection of methicillin resistance. - Also
- Reliable results were obtained at 37C, without
the addition of salt, without a specialised
medium and using two different inocula.
8Evaluation of a cefoxitin 30 µg disc on
Iso-Sensitest agar for detection of
methicillin-resistant Staphylococcus aureus.Skov
et al. J Antimicrob Chemother. 2003
Aug52(2)204-7.
- Skov et al. examined a difficult collection of
457 S. aureus strains including - 190 MRSA (including several defined PFGE types
and a number of low level resistant strains).
All MRSA were defined as mecA positive using PCR.
9Skov et al. (continued).
- Methods
- All strains were tested using
- Isosensitest agar
- A semi-confluent inoculum
- 30 µg cefoxitin disc
- Overnight incubation at 35-36C.
10Skov et al. (continued).
- SRGA Method tested in parallel
- All strains were tested using
- Isosensitest agar plus 5 horse blood.
- A confluent inoculum
- 1 µg oxacillin disc
- 24 h incubation at 30C.
11Skov et al. (continued).
- Results
- Using a zone diameter of lt 29 mm to define
resistance, the cefoxitin disc susceptibility
test showed a sensitivity of 100 and a
specificity of 99 . - The SRGA method using oxacillin (resistant lt 12
mm) showed a sensitivity of 78 and a
specificity of 99 .
12- Zone diameters of an oxacillin 1 µg disc the SRGA
method. A vertical line marks the present
interpretive zone diameter for susceptibility.
Black bars, mecA positive white bars, mecA
negative.
13 - Zone diameters of a cefoxitin 30 µg disc against
457 S. aureus A vertical line marks the proposed
interpretive zone diameter. Black bars, mecA
positive white bars, mecA negative.
14Significance of the study by Skov et al.
- The results show that susceptibility testing with
cefoxitin is much superior to the standard SRGA
method using oxacillin. - Also
- Reliable results were obtained at 35-36C using
Isosensitest agar with a semi-confluent inoculum. - These conditions are those recommended by the
BSAC Working Party for routine susceptibility
testing.
15Significance of the study by Skov et al.
- Disadvantages of the cefoxitin disc
susceptibility test - Marginal difference in zone diameter between
methicillin susceptible strains and some MRSA
strains. Accurate zone measurement is required. - Large zone sizes are produced by sensitive
strains that could potentially interfere with
other zones if multiple discs are tested on the
same plate.
16Evaluation of cefoxitin 5 and 10 µg discs for the
detection of methicillin resistance in
staphylococci (Skov et al. Journal of
Antimicrobial Chemotherapy 2005 55(2)157-161)
- Skov et al. examined a collection of 641 S.
aureus strains including 261 mecA negative and
380 mecA positive. - Test conditions
- Isosensitest agar and Mueller-Hinton agar.
- 10 µg and 5 µg cefoxitin discs.
- Semi-confluent inoculum.
- Incubation at 35 37C
- SRGA method using oxacillin also performed.
17Results Interpretive zone diameters for
Staphylococcus aureus for cefoxitin 5 and 10 µg
discs on ISA (Oxoid) and MH (BBL) and the
corresponding sensitivity and specificity
18Figure 1. Zone diameters against 641 S. aureus
using a semi-confluent inoculum and overnight
incubation in ambient air at 3537C. A vertical
line marks the proposed interpretive zone
diameter. Black bars, mecA-positive grey bars,
mecA-negative (a) 5 µg cefoxitin disc on
Iso-Sensitest agar (b) 10 µg cefoxitin disc on
Iso-Sensitest agar (c) 5 µg cefoxitin disc on
MuellerHinton agar (d) 10 µg cefoxitin disc on
MuellerHinton agar. n, no. of isolates with 6
mm zone.
19(No Transcript)
20Conclusions from the study by Skov et al.
- Cefoxitin 10 µg and 5 µg discs were both
successful for detection of MRSA. - Smaller zone diameters were produced with a high
proportion of MRSA giving no zone of inhibition. - Two mecA-positive strains isolated from Norway
could not be detected by any of the test methods
(including the oxacillin test). - There remains a marginal difference in zone
diameter between some strains of MRSA and MSSA.
21Evaluation of a 10 µg cefoxitin disc for the
detection of methicillin resistance in
Staphylococcus aureus by BSAC methodology.Andrews
et al. J Antimicrob Chemother. 2005
Sep56(3)599-600.
- In a study organised by the BSAC Working Party,
200 consecutive isolates of S. aureus (duplicates
from the same patient were excluded) were tested
in each of 5 different laboratories - City Hospital, Birmingham.
- St. Thomass Hospital, London
- Addenbrookes Hospital, Cambridge
- Royal Infirmary, Glasgow
- Freeman Hospital, Newcastle upon Tyne
22Andrews et al. (continued)
- Test conditions
- Isosensitest agar (Oxoid) depth 4 mm.
- Semi-confluent growth.
- 10 µg cefoxitin disc.
- Incubation at 34 36 C for 18 20 hours.
- All strains were also tested for the presence of
the mecA gene by PCR.
23Andrews et al. (continued) Results
- 328 strains of 1000 tested were MRSA as defined
by PCR. - 224 MRSA strains (68 ) showed no zone of
inhibition with a 10 µg cefoxitin disc. - 104 MRSA strains produced zone diameters between
719 mm. - A zone diameter breakpoint of 22 mm was chosen to
distinguish between MRSA and MSSA. - Using this breakpoint 2 strains of MSSA (out of
672) were falsely classified as methicillin
resistant.
24Zone diameter distribution for 1000 isolates of
S. aureus. A zone diameter breakpoint of 22 mm
was chosen to interpret susceptibility. Black
bars, mecA negative grey bars, mecA positive.
25Conclusions from the study by Andrews et al.
- As a result of this study (and other published
work) the BSAC now recommends the use of
cefoxitin as an option for the determination of
methicillin resistance in S. aureus. - Optimal test conditions Isosensitest agar,
Cefoxitin 10 µg discs, semi-confluent inoculum,
overnight incubation at 35C. - Interpretive criteria lt 22 mm Resistant.
26Coagulase-negative staphylococci (CNS) and
cefoxitin.
- The method now recommended by the BSAC for S.
aureus was employed by Skov et al. (2005) to
attempt to detect methicillin resistance in 344
strains of CNS - 132 were mecA negative and 212 were mecA
positive. - (Skov et al. Journal of Antimicrobial
Chemotherapy 2005 55(2)157-161)
27Skov et al. (2005)
- Test conditions
- (a) 5 µg cefoxitin disc on Iso-Sensitest agar
(b) 10 µg cefoxitin disc on Iso-Sensitest agar
(c) 5 µg cefoxitin disc on MuellerHinton agar
(d) 10 µg cefoxitin disc on MuellerHinton agar.
28Skov et al. (2005) Results
- None of the test conditions could reliably
classify CNS as either mecA-positive or
mecA-negative. - A scheme was proposed by the authors for reliable
interpretation of most isolates.
29Skov et al. (2005) continued
30J. Andrews et al. (2005) - unpublished
- 87 Coagulase-negative staphylococci tested using
BSAC recommendations for S. aureus versus
cefoxitin. - 51 mecA positive
- 36 mecA negative
31(No Transcript)
32Conclusions
- Cefoxitin disc susceptibility testing is a
reliable method for determination of methicillin
resistance in S. aureus. - Currently the BSAC Working Party are unable to
make recommendations for determination of
methicillin resistance in coagulase-negative
staphylococci using cefoxitin.