Guidelines for the Testing and Reporting of Antimicrobial Susceptibilities of Methicillin Resistant Staphylococcus aureus (MRSA) and Commentary on Methicillin Resistant Coagulase Negative Staphylococci (MR-CNS)

Canadian External Quality Assessment
Advisory Group for Antibiotic Resistance

September 1998

ISBN 0-662-27500-4
© 1998

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1.0 Introduction

These guidelines are produced under the auspices and authority of the Canadian External Quality Assessment - Advisory Group on Antibiotic Resistance (CEQA-AGAR). They represent a consensus of peer reviewed information and expert opinion on the most appropriate ways to test for and report antimicrobial susceptibility and resistance. Clinical situations and individual laboratory operations may necessitate some variations from these guidelines. These guidelines are strongly recommended for use by all laboratories providing information for national surveillance programs on antimicrobial resistance.

2.0 Preamble

In Canada, the first methicillin resistant Staphylococcus aureus (hereafter in this document referred to as MRSA) outbreak was reported in 1981. The Canadian Nosocomial Infection Surveillance Program (CNISP) showed a mean of 1.6 MRSA per 100 S. aureus isolates during 1995 and the first half of 1996 and a mean of 0.6 MRSA positive patients per 1,000 patient admissions ⁽¹⁾. This rate has been increasing in various parts of the country, and attention has been focused upon patient screening as a helpful early aid in limiting transmission.

This document, in an attempt to standardize the data which is being collected for national surveillance purposes, addresses which S. aureus isolates to test for methicillin resistance, how to test the isolates, and how to report isolates found to be methicillin resistant. Although these staphylococci are referred to as "methicillin-resistant", oxacillin is used in the laboratory to identify them. Routine testing is performed with oxacillin since it is more resistant to degradation in storage and more likely to detect heteroresistant strains. Resistance to oxacillin predicts resistance to all isoxasol penicillins.

3.0 Definitions

Classical MRSA strains are defined as having MICs to oxacillin 4 mg/L, however most strains have MICs 16 mg/L. The mecA gene which is responsible for this resistance is often associated in-vitro with resistance to all beta-lactam antibiotics. Susceptible strains have MIC's to oxacillin of < 4 mg/L. MRSA strains are frequently resistant to other classes of antibiotics.

Some S. aureus strains have borderline resistance to oxacillin with MICs between 4 and 16 mg/L and are mecA negative. The mechanism of resistance for these strains is believed to be hyper-production of beta-lactamase. The acronym BORSA has been used, meaning borderline oxacillin resistant S. aureus. Those organisms do not have the same infection control significance as MRSA.

4.0 Which S. aureus isolates to test for oxacillin resistance

4.1 S. aureus isolates which, according to individual laboratory protocols, are normally reported with susceptibilities are to be tested for oxacillin resistance.

4.2 MRSA screening surveillance can be done on request from patient specimens. Appropriate specimens for MRSA screening include: anterior nares (both nares on one swab), axillae (both axillae on one swab), perineum, drain sites, and open wounds. The anterior nares are often screened first, and other sites are screened next if the anterior nares are negative. This is because the initial screening of the anterior nares will pick up 80-90% of positive subjects. The specimens are plated directly onto a selective agar medium (e.g. Mannitol Salt Agar or Colistin-Naladixic Acid Agar) which is incubated at 35^o C for 48 hours. Any isolates recovered from the screening agar must be confirmed as true MRSA. Environmental cultures, except in exceptional situations, are rarely needed. However, if done, they can be handled by using the same screening agars as are used for patient specimens.

5.0 Testing S. aureus isolates for oxacillin resistance

5.1 Identification and speciation of S. aureus isolates must follow the established standard protocol. The protocol must address the recent observation that MRSA epidemics have been caused by a variant strain which is difficult to distinguish from coagulase negative staphylococci. For this reason, coagulase slide tests alone are not to be advocated because of false negative and false positive results. The gold standard remains the tube coagulase test which should be incubated for the FULL recommended time ⁽²⁾ (i.e.: 4 hours at 35^o C, followed by overnight at room temperature).

5.2 The ATCC control strains are:
Agar disk diffusion S. aureus ATCC 25923
MIC methods S. aureus ATCC 29213
Oxacillin agar screen No growth (susceptible) S. aureus ATCC 29213
Growth (resistant) S. aureus ATCC 43300.

5.3 Testing for oxacillin resistance in S. aureus must follow the most current NCCLS guidelines⁽³⁾, with strict attention to four critical factors:

• The inoculum must be prepared by a direct colony suspension method and is adjusted to match the 0.5 McFarland turbidity standard. It should be used immediately to spot or streak inoculate the screen plate. Any growth is considered resistant.

• The oxacillin screen is performed on Mueller Hinton agar containing 4% added NaCl with 6 mg/L oxacillin.

• Incubation temperature must be <= 35^o C.

• A full 24 hours of incubation is required.

• The ATCC control strains are specified in 5.2 above.

• Any growth on the plate is considered resistant.

The importance of the full 24 hours incubation cannot be overemphasized. Early readings may lead to false negative results; conversely, false positive tests arise after 24 hours leading to extra work and needless alarm. The media should be labelled with the time to avoid early or late readings.

5.4 Detection of susceptibility can be done by disc diffusion using a 1 µg oxacillin disk on Mueller Hinton without added NaCl at 35^o C. After 24 hours of incubation, the plate should be read using transmitted light. The zone diameters should be:

Resistant <= 10mm
Intermediate 11-12 mm
Susceptible 13 mm

Regardless of zone size any discernable growth within the zone of inhibition is indicative of resistance.

5.5 Automated identification and susceptibility methods are unreliable for the detection of MRSA. All S. aureus isolates should be tested on oxacillin plates. All potentially oxacillin resistant isolates should be confirmed by an oxacillin MIC method (E-Test^®, agar or broth dilution methods).

5.6 Strains with MICs between 4 and 16 mg/L should be sent to a reference laboratory for mecA detection.

6.0 Reporting S. aureus isolates found to be oxacillin resistant

6.1 The Medical Microbiologist/Laboratory Physician and/or the Infection Control Practitioner should be notified about any S. aureus isolate which is recovered from the oxacillin screening plate or which demonstrates oxacillin resistance by disk diffusion testing.

6.2 S. aureus with confirmed resistance to oxacillin are considered to be resistant to all other beta-lactam antibiotics including penicillins, cephalosporins, cephems, carbapenems, and beta-lactam/beta-lactamase inhibitor combinations. Regardless of the in vitro susceptibilities, the microbiology report should reflect this resistance profile.

6.3 MRSA strains should be reported as required by provincial legislation or agreement.

6.4 Organisms sent to a reference laboratory should be stocked by the referring laboratory until conformation is received from the reference laboratory. The reference laboratory should stock the isolates for at least six months, or as required by provincial or local regulation.

6.5 For external/national surveillance reporting purposes, for isolates confirmed to be MRSA, report one isolate per patient.

7.0 Summary

Isolates of MRSA may be obtained from clinical specimens or patient or environmental specimens taken for the purposes of surveillance. These isolates must be confirmed as oxacillin resistant. Increasingly this also includes demonstration of genetic determinant of resistance (mecA). Reporting of these isolates is determined internally at each institution. For those institutions participating in external surveillance, only one isolate per patient should be reported.

8.0 References

1. Simor, A., et al. The Canadian Nosocomial Infection Surveillance Program: Results of the first 18 months of Surveillance for Methicillin Resistant Staphylococcus aureus in Canadian Hospitals. CCDR, 3-26, 1997.

2. Murray, Patarick R. et al . (eds). Manual of Clinical Microbiology. 6th edition, ASM Press, Washington, D.C. 1995.

3. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing; Eighth Informational Supplement. M100 S-8 Vol. 17, No. 1 and No. 2. Villanova: National Committee for Clinical Laboratory Standards, January 1998.

4. Canadian Association of Medical Microbiologists, B.C. Chapter, Recommendations: Laboratory identification and susceptibility testing of Methicillin Resistant Staphylococcus aureus, BCCAMM focus group, Final Draft. Jan 1998.

Commentary on Susceptibility Testing on Methicillin Resistant Coagulase Negative Staphylococci

Testing of coagulase negative staphylococci (CNS) for antibiotic resistance should only be done when the isolate is considered to be a likely pathogen in an infective process. When the decision has been made to perform susceptibility testing, the following methods should be followed for determining oxacillin resistance. Resistance to methicillin is based on carriage of mecA gene which may not be phenotypically expressed with the same efficiency in coagulase negative staphylococci, as it is in Staphylococcus aureus.

1. The 6 mg/L oxacillin agar screen plate is not suitable for detection of MR-CNS. Strains that appear susceptible by the 6 mg/L oxacillin agar screen plate may be mecA positive and clinically resistant to beta-lactam antibiotics.

2. Disk diffusion with a 1 µg oxacillin disk on Mueller Hinton agar without added salt; inoculation from a direct colony suspension to a 0.5 McFarland standard, incubation for a full 24 hr (48 hr if resistance as defined below is not observed in 24 hr) at 35^o C. In order to identify all isolates carrying mecA, plates should be read as per MRSA (see 5.4 above) . Zone diameter criteria are:

Resistant - <= 17 mm
Susceptible - >= 18 mm

3. CNS can also be tested for oxacillin resistance using an MIC method in cation-adjusted Mueller Hinton broth. Breakpoints for MR-CNS are lower than for MRSA:

Resistant - >= 0.5 mg/L
Susceptible - <= 0.25 mg/L

4. Determination of oxacillin resistance in strains of S. lugdunensus, S. schlieferi, and S. saprophyticus appears to correlate better using the MIC and zone diameter breakpoints for S. aureus (see 5.4 above). Until further studies are available, these species should be tested for oxacillin resistance using S. aureus criteria.

5. When reporting results, strains of MR-CNS, like S. aureus, should be considered resistant to all beta-lactam antibiotics, including penicillins, cephalosporins, cephamycins, carbapenems, and beta-lactam/beta-lactamase inhibitor combinations.

6. MR-CNS are not generally considered an infection control threat, and notification practices considered standard for MRSA are not necessary for MR-CNS.

7. There is no need to forward isolates of MR-CNS to a reference laboratory for mecA detection.

References for MR-CNS

1. Marshall SA, Wilke WW, Pfaller MA, Jones RN. 1998. Staphylococcus aureus and coagulase-negative staphylococci from blood stream infections: frequency of occurrence, antimicrobial susceptibility and molecular (mecA) characterization of oxacillin resistance in the SCOPE program. Diagn. Microbiol. Infect. Dis. 30: 205-214.

2. Ramotar K, Bobrowska M, Jessamine P, Toye, B. 1998. Detection of methicillin resistance in coagulase-negative staphylococci initially reported as methicillin susceptible using automated methods. Diagn. Microbiol. Infect. Dis. 30: 267-273.

[CEQA-AGAR]