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Methicillin resistance in staphylococci

15 min

Skills you'll build3 skills
  • Detect methicillin resistance in staphylococci with a cefoxitin screen
  • Recall the altered penicillin-binding protein encoded by mecA
  • Name what a negative mecA, nitrocefin, or other targeted test cannot detect

Try first

Try first

A Staphylococcus aureus from a blood culture gives a cefoxitin disk zone of 19 mm at 18 hours. The laboratory's cefoxitin criteria for S. aureus are susceptible 22 mm or more and resistant 21 mm or less. What is reported?

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

PBP2a and methicillin resistance

Methicillin resistance in staphylococci is caused by PBP2a, a penicillin-binding protein with low affinity for beta-lactams. The mecA gene encodes it, and the rarer mecC encodes a divergent homologue, PBP2c. PBP2a makes the isolate resistant to nearly every beta-lactam, including combinations with a beta-lactamase inhibitor. The cephalosporins made for MRSA, such as ceftaroline, are the exception.1,2

Expression can be heterogeneous. A small resistant subpopulation can make an isolate appear susceptible, so incubation above 35 °C or an early read can miss the resistance.2

Choosing the test by species

The Clinical and Laboratory Standards Institute (CLSI) sets the valid tests and their reading times species by species.2,3

TestValid forRead at
Cefoxitin diskS. aureus, S. lugdunensis16 to 18 hours
Cefoxitin diskS. epidermidis and other listed coagulase-negative staphylococci24 hours
Cefoxitin MICS. aureus, S. lugdunensis only16 to 20 hours
Oxacillin MICAll staphylococci, with species-specific breakpoints24 hours

Cefoxitin induces mecA strongly and gives a clearer endpoint than oxacillin.1 A PBP2a latex or lateral-flow test on a colony detects the protein directly and gives a rapid confirmation.2 S. pseudintermedius, S. schleiferi and S. coagulans are not covered by the cefoxitin disk's validation, and they are tested with oxacillin.1,2

Limits of a targeted test

A targeted test detects only its own target:

  • A mecA PCR misses mecC.
  • Some PBP2a reagents miss the mecC protein, PBP2c, even after induction.4
  • A negative nitrocefin test in Haemophilus influenzae misses beta-lactamase-negative, ampicillin-resistant (BLNAR) strains, whose resistance is caused by altered PBPs.2

The phenotypic result determines the category when a targeted test is negative. A resistant genotype is also reported when the phenotype appears susceptible, because some isolates have mecA and still test oxacillin susceptible. An S. aureus with a genotype and phenotype that cannot be reconciled is reported resistant.2

CLSI makes one exception for staphylococci other than S. aureus, S. lugdunensis, S. epidermidis, S. pseudintermedius and S. schleiferi. Their oxacillin MIC breakpoints can overcall resistance. An isolate from a serious infection with an oxacillin MIC of 1 or 2 µg/mL may be tested for mecA or PBP2a, and an isolate that tests negative is reported oxacillin susceptible.2,5

Follow the bench demonstration

Sources for this step
References
  1. Centers for Disease Control and Prevention. Laboratory testing for methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA). Reviewed June 27, 2025. Accessed September 27, 2026. https://www.cdc.gov/mrsa/php/laboratories/index.html
  2. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 36th ed. CLSI supplement M100. CLSI; 2026. Accessed September 27, 2026. https://clsi.org/shop/standards/m100/
  3. Clinical and Laboratory Standards Institute. M100, 30th ed., combined corrections. September 14, 2020. Accessed September 27, 2026. https://clsi.org/media/3824/m100ed30_combined_correction_notice_20200914_web.pdf
  4. Dupieux C, Mouton W, André C, et al. Performance of the revised version of an immunochromatographic assay for detection of mecA- and mecC-mediated methicillin resistance in staphylococci. J Clin Microbiol. 2019;58(1):e01346-19. doi:10.1128/jcm.01346-19
  5. Clinical and Laboratory Standards Institute. AST news update June 2022: case studies. Published June 21, 2022. Accessed October 23, 2026. https://clsi.org/about/press-releases/ast-news-update-june-2022-case-studies/

Watch one

Read each decision and its explanation.

A Staphylococcus epidermidis grows from two sets of blood cultures in a patient with a prosthetic hip. The cefoxitin disk was read at 18 hours with a zone of 27 mm. The laboratory's cefoxitin criteria for S. epidermidis are susceptible 25 mm or more and resistant 24 mm or less.

What is reported for oxacillin?

  1. Confirm the species. S. epidermidis is covered by the cefoxitin disk's validation.

    The species determines which test and which interpretive criteria apply.

  2. Check the reading time. S. epidermidis is read at 24 hours, so the 18-hour zone is not a reportable result.

    Coagulase-negative staphylococci need the full incubation for slow-growing resistant subpopulations to appear.

  3. Read at 24 hours. Fine growth now extends into the zone, which measures 23 mm.

    Heterogeneous resistant growth can appear within the zone late in the incubation.

  4. Compare with the criteria. 23 mm is 24 mm or less, so the isolate is resistant.

    The cefoxitin disk has only two categories.

  5. Report oxacillin resistant. The other beta-lactams follow the laboratory's rule for methicillin-resistant staphylococci.

    Cefoxitin is the surrogate for oxacillin, and PBP2a confers resistance to nearly every beta-lactam.

Oxacillin (methicillin) resistant, from the 24-hour cefoxitin zone of 23 mm.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

Why is cefoxitin useful in phenotypic testing for methicillin resistance in S. aureus?

Cefoxitin supports detection of methicillin resistance. Vancomycin susceptibility needs its own appropriate method.

This is a growth-based test. A nucleic acid assay directly detects its specified genetic target.

Cefoxitin is a strong mecA inducer and provides endpoints that are easier to interpret than oxacillin in this application.

Hint
  1. Cefoxitin is a surrogate. Consider which drug it is reported as.
  2. The disk test measures growth on a plate. Consider what a growth-based test can and cannot show.

Review Staphylococcal methicillin resistance

Problem 2 of 3 · Hints available

A blood isolate of Haemophilus influenzae is β-lactamase-negative by nitrocefin. What does this mean for ampicillin?

Incorrect. BLNAR strains test nitrocefin negative and ampicillin resistant, and CLSI recommends ampicillin testing for blood and CSF isolates, so the test is still performed.

Incorrect. TEM-1 and ROB-1 are what a positive nitrocefin screen detects. This screen is negative, so any ampicillin resistance would come from altered penicillin-binding proteins.

Correct. β-Lactamase-negative, ampicillin-resistant (BLNAR) strains resist through altered penicillin-binding proteins that nitrocefin cannot detect. CLSI recommends ampicillin testing for blood and CSF isolates of H. influenzae, so this blood isolate still receives full susceptibility testing.

Hint
  1. Nitrocefin detects an enzyme. Consider whether an enzyme is the only mechanism of ampicillin resistance.
  2. Recall another mechanism of penicillin resistance.

Review HACEK organisms and Haemophilus identification

Problem 3 of 3 · On your own

An S. aureus from a dairy farmer's wound gives a cefoxitin disk zone of 16 mm, resistant by the laboratory's criteria. A mecA PCR on the colony is negative, and so is a PBP2a latex test. What is reported for oxacillin?

The PCR detects mecA and the latex detects mecA-encoded PBP2a. They can miss mecC, and the cefoxitin result shows resistance.

Called an isolate susceptible from a negative targeted test

A targeted test detects only its targets. A mecA assay misses mecC, and a negative nitrocefin screen misses beta-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus with altered PBPs. Reporting susceptibility from the negative test can release a resistant isolate, so report the category from the phenotypic result.

The phenotypic result determines the category. A strain carrying mecC, a rarer gene linked to livestock and wildlife, fits a resistant cefoxitin zone with a negative mecA test.

The cefoxitin result is valid and reportable now. Sequencing can explain the mechanism later.

Review Staphylococcal methicillin resistance

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • A Staphylococcus pseudintermedius grows from a dog-bite wound on a veterinary technician's hand.
  • The cefoxitin disk on the routine panel shows a large zone.
  • The laboratory's oxacillin MIC breakpoints for S. pseudintermedius are susceptible 0.25 µg/mL or less and resistant 0.5 µg/mL or more.
  • The oxacillin MIC, set up next, reads 2 µg/mL at 24 hours.
  • The ward calls to ask whether amoxicillin-clavulanate can be reported susceptible, since clavulanate inhibits beta-lactamase.
Decision 1 of 2

What do you do with the cefoxitin zone?

The cefoxitin disk is not validated for S. pseudintermedius. A large zone can occur with mecA-mediated resistance.

Read a cefoxitin disk on S. pseudintermedius

The cefoxitin disk is validated for S. aureus, S. lugdunensis, S. epidermidis, and other listed coagulase-negative species. S. pseudintermedius, S. schleiferi, and S. coagulans fall outside that validation, so a cefoxitin zone on them can report a methicillin-resistant isolate as susceptible.

S. pseudintermedius is not covered by the cefoxitin disk's validation. The oxacillin MIC with its species breakpoints determines the category.

Criteria for one species do not apply to another. The cefoxitin disk has no validated criteria for S. pseudintermedius.

Read a cefoxitin disk on S. pseudintermedius

The cefoxitin disk is validated for S. aureus, S. lugdunensis, S. epidermidis, and other listed coagulase-negative species. S. pseudintermedius, S. schleiferi, and S. coagulans fall outside that validation, so a cefoxitin zone on them can report a methicillin-resistant isolate as susceptible.

Review Staphylococcal methicillin resistance

Decision 2 of 2

The oxacillin MIC of 2 µg/mL is resistant. How is amoxicillin-clavulanate reported?

Clavulanate inhibits beta-lactamases. PBP2a is an altered target protein, and clavulanate has no effect on it.

Confused the mecA product with a beta-lactamase

mecA encodes PBP2a, an altered penicillin-binding protein with low beta-lactam affinity. Calling it a beta-lactamase suggests an inhibitor combination would restore activity. PBP2a confers resistance to nearly all beta-lactams, inhibitor combinations included, except anti-MRSA cephalosporins such as ceftaroline.

mecA encodes PBP2a, which confers resistance to nearly every beta-lactam, including inhibitor combinations.

An oxacillin-resistant staphylococcus is reported resistant to beta-lactam-inhibitor combinations, regardless of what a separate test shows.

Review Staphylococcal methicillin resistance

The interpretation depends on the species. S. pseudintermedius is not covered by the cefoxitin disk's validation, so the large zone is not reportable. The oxacillin MIC shows PBP2a-mediated resistance, and clavulanate has no effect on PBP2a.

For reference

Sources checked