How Far Do We Have to Go to Report Methicillin-Resistant Staphylococcus aureus (MRSA)?

We recently had a case that made me stop to think about the lengths we sometimes have to go to understand what the bacteria are trying to teach us. 

The Issue

Not only are there several methods for the detection of oxacillin resistance in staphylococci (1,2,3), but many laboratories employ a variety of those methods (image 1). This can be a blessing…or a curse. Blessing: alternate methods can be used to referee discordant results. Curse: testing additional methods may cause more confusion. To quote Frank Lloyd Wright, “Less is more only when more is too much.”

The mecA gene encodes for the alternate Penicillin Binding Protein 2a (PBP2a), which confers resistance to oxacillin. In recent years, cefoxitin has become the preferred surrogate for oxacillin to detect methicillin resistance (1, 2). Moreover, as molecular methods become more widely adopted, we open ourselves up to scenarios where the genotype does not match the phenotype. Depending on how your laboratory reports these results, it can be confusing to clinicians. All they want to know is, “Is this MRSA, or not?”

When molecular and culture results do not agree

Many laboratories have turned to polymerase chain reaction (PCR) assays to detect MRSA because of its rapid time to result and its superior sensitivity (compared to AST and PBP2a testing). Recent advances in technology allow for the detection of MRSA direct from positive blood cultures. Rapid organism identification and detection of resistance markers greatly improve patient care by reducing the time to appropriate antimicrobial therapy. However, the problem occurs when the PCR result does not match the PBP2a and/or AST results.

In our laboratory, when a blood culture flags positive the bottles are removed from the automated instrument, a Gram stain is performed, the specimen is subcultured (blood, chocolate, and MacConkey agars) and analyzed (if appropriate) by a multiplex PCR. This workflow allows us to report organism identifications on the majority of our blood cultures on day one of the culture flagging positive (image 1). Final AST results are usually reported by day three. Of note, additional steps are added depending on the Gram stain result. For example, if the Gram stain reveals gram-positive cocci in clusters (GPCL) suggestive of Staphylococcus, then we will also drop a cefoxitin disk onto the blood agar plate.

For this particular case, on day one the Gram stain revealed GPCL and the PCR assay detected S. aureus and mecA, which indicated that the patient had MRSA bacteremia. Nothing out of the ordinary. On day two, we confirmed the culture was S. aureus, but the PBP2a result was negative. Unfortunately, the cefoxitin disk was missed when the culture was originally subcultured (image 2A). Normally, we perform PBP2a testing from cefoxitin-induced growth around the zone of inhibition, but that did not occur in this case. Because the PBP2a and PCR results were discordant, the blood culture was subcultured again to include the cefoxitin disk (image 2B). 

The Solution

Image 2A shows a homogenous culture of S. aureus. Under examination no differences could be detected among the colonies; it appears to be a pure culture. Image 2B shows that the culture is actually a heterogeneous culture with both MRSA and MSSA. There are two zones of inhibition around the cefoxitin disk. The white circle highlights the cefoxitin-resistant population and the blue circle highlights the susceptible population. The isolated colonies in the third quadrant look identical; one cannot distinguish between the two populations. This example displays the heterogeneous nature of S. aureus in terms of resistance, or rather its “heteroresistance” to oxacillin and the complexity involved in detecting MRSA.

For those that use phenotypic assays, the use of a cefoxitin disk can help improve your detection rates. Because cefoxitin is both a surrogate for oxacillin resistance and a strong beta-lactamase inducer, it is the perfect aid to enhance MRSA detection (image 2). For this reason, our laboratory implemented the addition of the cefoxitin disk to positive blood cultures with GPLCs. Because we had a few false-negative PBP2a MRSA cases, we decided to start performing PBP2a testing on cefoxitin-induced growth. This intervention has helped two-fold. First, it has seemingly reduced the number of false-negative results. Second, the zone of inhibition functions as an internal control. If the zone is small or non-existent, then it seems obvious that isolate should be resistant. If the zone is large, then we would expect the isolate to be susceptible. Moreover, if we see a double zone as in image 2A, then the culture is likely mixed. [As an aside, we once had a case where the mecA gene detected was from a coagulase-negative Staphylococcus and not the S. aureus (MSSA) that was also present in the culture.]

mrsa1
Image 1.  S. aureus workflow. From the time the specimen comes into the laboratory, it can take anywhere from one to three days before a provider knows if their patient has methicillin-susceptible S. aureus (MSSA) or MRSA.  MRSA isolates are multidrug-resistant, which require broad-spectrum antibiotics, such as vancomycin (3). For patients with MSSA, narrow-spectrum beta-lactam antibiotics, such as nafcillin or cefazolin are not only the first-line therapy, but superior in terms of outcomes compared to vancomycin (4). Also, patients with MRSA may be put in isolation depending on your institution’s infection control protocol. More importantly, delay of appropriate antibiotics can significantly increase mortality, especially in the case of sepsis (5).
mrsa2
Image 2. Cefoxitin induces methicillin resistance. A) A positive blood culture was subcultured to blood agar (no cefoxitin disk). B) The same positive blood culture was subcultured with a cefoxitin disk (30 mg). FOX= cefoxitin. Recall that cefoxitin is a surrogate for oxacillin, therefore resistance to either (oxacillin or cefoxitin) denotes resistance to methicillin.

The Conclusion

Depending on the S. aureus isolate, the detection of methicillin resistance can be a challenge regardless of which method(s) your laboratory utilizes. Fortunately, there is a clear-cut solution. CLSI recommends that isolates should be reported as MRSA if oxacillin resistance is detected by any method (1, 2).

As you saw from our case, the phenotype did actually match the genotype. A combination of heteroresistance and human error (un-induced growth for PBP2a) led us astray. As you read this, you may be thinking, “why did they do all of that?” Good question! We felt that this was a good case to teach and/or learn. “Smart people learn from their mistakes. Geniuses learn from others.” If this can help others learn, then it is all worth it. My gift to you. Merry Christmas!

References

  1. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard- tenth edition. CLSI document M07-A10. Wayne, PA: Clinical and Laboratory Standards Institute; 2015.
  2. Performance standards for antimicrobial susceptibility testing. CLSI document M100-S27. Wayne, PA: Clinical and Laboratory Standards Institute; 2017.
  3. Centers for Disease Control and Prevention (CDC). Methicillin-resistant Staphylococcus aureus (MRSA). https://www.cdc.gov/mrsa/lab/index.html. Accessed December 18, 2017.
  4. Schweizer, M.L., Furano, J.P., Harrris, A.D., Johnson, J.K., Shardell, M.D., McGregor, J.C., Thom, K.A., Cosgrove, S.E., Sakoulas, G. and Perencevich, E.N. (2011). Comparative effectiveness of nafcillin or cefazolin versus vancomycin in methicillin-susceptible Staphylococcus aureus BMC Infect. Dis. 11:279. doi: 10.1186/1471-2334-11-279
  5. Kumar, A., Roberts, D., Wood, K.E., Light, B., Parrillo, J.E., Sharma, S., Suppes, R., Feinstein, D., Zanotti, S., Taiberg, L., Gurka, D., Kumar, J. Cheang, M. 2006. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Care Med. 34:1589-1596. doi: 10. 1097/01.CCM.0000217961.75225.E9

 

Martinez Headshot-small 2017

-Raquel Martinez, PhD, D(ABMM), was named an ASCP 40 Under Forty TOP FIVE honoree for 2017. She is one of two System Directors of Clinical and Molecular Microbiology at Geisinger Health System in Danville, Pennsylvania. Her research interests focus on infectious disease diagnostics, specifically rapid molecular technologies for the detection of bloodstream and respiratory virus infections, and antimicrobial resistance, with the overall goal to improve patient outcomes.

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