Transcript The Use of Cefoxitin for the Determination of Methicillin

The Use of Cefoxitin for the
Determination of Methicillin
Resistance in Staphylococci
John D. Perry
Microbiology Department
Freeman Hospital
Newcastle upon Tyne
BSAC Working Party
recommendations for detection of
methicillin resistance (2004):
Inoculum: Semi confluent
Medium: Columbia agar plus 2 % salt.
Incubation conditions: 30°C
Discs: Oxacillin 1 µg or methicillin 5 µg
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.
Felten et al:
Evaluation 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 Aug;40(8):2766-71.
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 37°C using high and low
inocula.
Oxacillin disc testing was performed in parallel
using CLSI and CASFM methodologies.
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 < 27 mm = MRSA.
Disc testing using oxacillin showed a sensitivity
of 95.2 – 96.4 % depending on the inoculum
used. Specificity was 100 %.
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 37°C, without
the addition of salt, without a specialised
medium and using two different inocula.
Evaluation of a cefoxitin 30 µg disc on IsoSensitest agar for detection of methicillinresistant Staphylococcus aureus.
Skov et al. J Antimicrob Chemother. 2003 Aug;52(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.
Skov et al. (continued).
Methods:
All strains were tested using:
Isosensitest agar
A semi-confluent inoculum
30 µg cefoxitin disc
Overnight incubation at 35-36°C.
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 30°C.
Skov et al. (continued).
Results:
Using a zone diameter of < 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 <
12 mm) showed a sensitivity of 78 % and a
specificity of 99 %.
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.
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.
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 35-36°C using
Isosensitest agar with a semi-confluent inoculum.
These conditions are those recommended by the
BSAC Working Party for routine susceptibility
testing.
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.
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 – 37°C
SRGA method using oxacillin also performed.
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
Agar Disc
Interpretive zone diameter (mm) Sensitivity (%) Specificity (%
ISA
cefoxitin 5 µg
R < 14
99.5
98.1
ISA
cefoxitin 10 µg R < 22
99.5
98.1
MH
cefoxitin 5 µg
R < 12
99.7
98.1
MH
cefoxitin 10 µg R < 18
99.5
98.9
ISA
oxacillin 1 µg
82.3
100
R < 12
Figure 1. Zone diameters against 641 S. aureus
using a semi-confluent inoculum and overnight
incubation in ambient air at 35–37°C. 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 Mueller–Hinton agar;
(d) 10 µg cefoxitin disc on Mueller–Hinton agar.
n, no. of isolates with 6 mm zone.
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.
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 Sep;56(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. Thomas’s Hospital, London
•Addenbrookes Hospital, Cambridge
•Royal Infirmary, Glasgow
•Freeman Hospital, Newcastle upon Tyne
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.
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
7–19 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.
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.
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 35°C.
Interpretive criteria: < 22 mm = Resistant.
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)
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 Mueller–Hinton agar;
(d) 10 µg cefoxitin disc on Mueller–Hinton agar.
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.
Skov et al. (2005) – continued:
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
CNS tested on ISA cefoxitin 10 ug discs incubation @ 35'C
25
Number
20
15
Mec A -ve
MecA +ve
10
5
0
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 33 34 35 36
Zone diameter (mm)
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.