The%20Use%20of%20Cefoxitin%20for%20the%20Determination%20of%20Methicillin%20Resistance%20in%20Staphylococci - PowerPoint PPT Presentation

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The Use of Cefoxitin for the Determination of Methicillin Resistance in Staphylococci John D. Perry Microbiology Department Freeman Hospital Newcastle upon Tyne – PowerPoint PPT presentation

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Title: The%20Use%20of%20Cefoxitin%20for%20the%20Determination%20of%20Methicillin%20Resistance%20in%20Staphylococci


1
The Use of Cefoxitin for the Determination of
Methicillin Resistance in Staphylococci
  • John D. Perry
  • Microbiology Department
  • Freeman Hospital
  • Newcastle upon Tyne

2
BSAC 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

3
BSAC 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.

4
Felten 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.

6
Felten 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 .

7
Significance 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.

8
Evaluation 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.

9
Skov et al. (continued).
  • Methods
  • All strains were tested using
  • Isosensitest agar
  • A semi-confluent inoculum
  • 30 µg cefoxitin disc
  • Overnight incubation at 35-36C.

10
Skov 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.

11
Skov 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.

14
Significance 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.

15
Significance 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.

16
Evaluation 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.

17
Results 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
18
Figure 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
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20
Conclusions 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.

21
Evaluation 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

22
Andrews 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.

23
Andrews 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.

24
Zone 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.
25
Conclusions 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.

26
Coagulase-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)

27
Skov 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.

28
Skov 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.

29
Skov et al. (2005) continued
30
J. 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
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32
Conclusions
  • 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.
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