LETTER
Detection of methicillin resistance in staphylococcal isolates is crucial to quickly optimize treatment in cases of severe staphylococcal infections. Rapid genetic or phenotypic tests have been developed to detect isolates harboring the mecA gene, which is responsible for methicillin resistance related to additional penicillin-binding protein 2a (PBP2a) expression. The discovery in 2011 of a mecA variant, named mecC, encoding PBP2c and showing only 70% nucleotide sequence homology, was a real challenge for laboratories, since most of the tests targeting mecA failed to detect mecC (1). The first version of the immunochromatographic assay developed by Alere (PBP2a culture colony test; Alere, Scarborough, ME), based on blue-colored monoclonal antibodies targeting PBP2a, has been widely evaluated. It demonstrated 100% specificity and sensitivity for mecA-positive Staphylococcus aureus strains but displayed lower sensitivity for Staphylococcus non-aureus (SNA) species such as Staphylococcus epidermidis (2–4). For mecC-positive isolates, this assay revealed a lack of sensitivity unless PBP2c expression was induced by 18-h culture around a cefoxitin disk, which increased the sensitivity from 8.7% to 100% (5). Alere recently updated this test, whose intended use is still the detection of PBP2a from S. aureus isolates, in order to allow storage at room temperature and changed the antibodies immobilized on the nitrocellulose membrane, which are now recombinant monoclonal antibody fragments with a conjugate labeled red (6). Here, we evaluated the revised version of the assay, the PBP2a SA culture colony test (SACCT) (Alere), using a collection of both S. aureus and SNA isolates, in order to determine whether the technical changes, particularly the use of antibody fragments, affected the performance of the immunochromatographic test.
A large collection of methicillin-susceptible S. aureus and methicillin-resistant S. aureus (MRSA) isolates (n = 83) belonging to more than 50 different clones and methicillin-susceptible and methicillin-resistant SNA isolates (n = 122) belonging to 14 different species was tested (see Table 1 for details). The presence of mecA and mecC was confirmed using PCR (7, 8). All S. aureus isolates had been previously characterized by spa typing, multilocus sequence typing, and/or microarray analysis (Alere Technologies, Jena, Germany) to ensure diversity of the tested strains, which were representative of the most prevalent clones circulating in Europe. Isolates were grown for 18 to 24 h on Columbia sheep blood agar plates (bioMérieux, La-Balme-les-Grottes, France) and then were tested directly using the PBP2a SACCT, according to the manufacturer’s instructions. The assay was also performed after PBP2a/PBP2c induction using colonies harvested around a cefoxitin disk (30 μg). The testing was performed by a single operator, without replicate, except in cases of discrepancies between the results of the mecA/mecC PCR and immunochromatographic assays.
TABLE 1.
Performance of the immunochromatographic assay PBP2a SACCT (Alere) with a large collection of staphylococcal isolates
| Species | No. of isolates tested | Genetic background(s) includeda | No. of isolates |
|||
|---|---|---|---|---|---|---|
| Without induction |
After cefoxitin induction |
|||||
| Positive result | Negative result | Positive result | Negative result | |||
| mecA- and mecC-negative isolates (n = 24) | ||||||
| Staphylococcus aureus | 10 | CC5, CC8, CC15, CC30, CC45, CC49, CC101, CC182, CC398, and CC1021 | 0 | 10 | 0 | 10 |
| Staphylococcus argenteus | 1 | CC2250/CC2277 | 0 | 1 | 0 | 1 |
| Staphylococcus schweitzeri | 1 | ST2022 | 0 | 1 | 0 | 1 |
| Staphylococcus epidermidis | 1 | ND | 0 | 1 | 0 | 1 |
| Staphylococcus haemolyticus | 1 | ND | 0 | 1 | 0 | 1 |
| Staphylococcus hominis | 1 | ND | 0 | 1 | 0 | 1 |
| Staphylococcus capitis | 1 | ND | 0 | 1 | 0 | 1 |
| Staphylococcus pseudintermedius | 1 | ND | 0 | 1 | 0 | 1 |
| Staphylococcus cohnii | 1 | ND | 0 | 1 | 0 | 1 |
| Staphylococcus saprophyticus | 1 | ND | 0 | 1 | 0 | 1 |
| Staphylococcus warneri | 1 | ND | 0 | 1 | 0 | 1 |
| Staphylococcus sciuri | 1 | ND | 0 | 1 | 0 | 1 |
| Staphylococcus simulans | 1 | ND | 0 | 1 | 0 | 1 |
| Staphylococcus lugdunensis | 1 | ND | 0 | 1 | 0 | 1 |
| Staphylococcus caprae | 1 | ND | 0 | 1 | 0 | 1 |
| mecA-positive isolates (S. aureus [n = 63] and other species [n = 108]) | ||||||
| Staphylococcus aureus | 63 | 43 different clonesb | 63 | 0 | 63 | 0 |
| Staphylococcus argenteus | 2 | ST1850-MRSA-IV and CC2250/CC2277-MRSA-IV | 2 | 0 | 2 | 0 |
| Staphylococcus schweitzeri | 0 | ND | 0 | 0 | 0 | 0 |
| Staphylococcus pseudintermedius | 15 | ND | 15 | 0 | 15 | 0 |
| Staphylococcus epidermidis | 11 | ND | 11 | 0 | 11 | 0 |
| Staphylococcus haemolyticus | 11 | ND | 11 | 0 | 11 | 0 |
| Staphylococcus hominis | 11 | ND | 11 | 0 | 11 | 0 |
| Staphylococcus capitis | 11 | ND | 11 | 0 | 11 | 0 |
| Staphylococcus cohnii | 11 | ND | 11 | 0 | 11 | 0 |
| Staphylococcus saprophyticus | 11 | ND | 10 | 1 | 11 | 0 |
| Staphylococcus warneri | 5 | ND | 5 | 0 | 5 | 0 |
| Staphylococcus sciuri | 5 | ND | 5 | 0 | 5 | 0 |
| Staphylococcus simulans | 5 | ND | 5 | 0 | 5 | 0 |
| Staphylococcus lugdunensis | 9 | ND | 9 | 0 | 9 | 0 |
| Staphylococcus caprae | 1 | ND | 1 | 0 | 1 | 0 |
| mecC-positive S. aureus isolates (n = 10) | ||||||
| Staphylococcus aureus | 10 | CC130, CC425, and CC1943 (corresponding to spa-types t742, t843, t978, t2345, t6220, and t6300) | 0 | 10 | 0 | 10 |
CC, clonal complex; ST, sequence type; ND, not determined.
The mecA-positive S. aureus isolates belonged to the following clones: CC1-MRSA-IV, WA MRSA-1/45; CC1-MRSA-IV, WA MRSA-1/57; CC1-MRSA-V; CC5-MRSA-II, Rhine-Hesse epidemic MRSA (EMRSA)/UK-EMRSA-3; CC5-MRSA-IV, pediatric clone; CC5-MRSA-V, WA MRSA-81/85/86/123; CC8-MRSA-IV, Lyon clone; CC8-MRSA-VIII; CC22-MRSA-V; CC30-MRSA-IV, Southwest Pacific clone; CC45-MRSA-IV, Berlin EMRSA; CC59-MRSA-IV, USA1000; CC80-MRSA-IV, European community-acquired MRSA clone; CC88-MRSA-IV, WA MRSA-2; CC88-MRSA-V; CC97-MRSA-IV, WA MRSA-54/63; CC97-MRSA-V; CC121-MRSA-IV; CC398-MRSA-IV; CC398-MRSA-IX; CC398-MRSA-X; CC779-MRSA-IV, WA MRSA-100; ST5-MRSA-I, Geraldine clone; ST5-MRSA-VII; ST6-MRSA-IV, WA MRSA-51; ST8-MRSA-IV, arginine catabolic mobile element (ACME)-negative variant of USA300; ST22-MRSA-IV, Barnim/UK-EMRSA-15; ST36-MRSA-II, UK-EMRSA-16; ST59/952-MRSA-V(T), Taiwan clone; ST59-MRSA-V; ST72-MRSA-IV, USA700; ST87-MRSA-IV, WA MRSA-24; ST93-MRSA-IV, Queensland clone; ST125-MRSA-IV; ST146-MRSA-IV; ST188-MRSA-IV; ST228-MRSA-I, South German EMRSA/Italian clone; ST239-MRSA-III, Vienna/Hungarian/Brazilian clone; ST241; ST377; ST398-MRSA-V; ST573/772-MRSA-V; and ST772-MRSA-V, Bengal Bay clone.
The 24 methicillin-susceptible isolates yielded negative results for PBP2a detection. Regarding the mecA-positive isolates, all MRSA isolates tested positive without induction of PBP2a expression. Conversely, none of the mecC-positive S. aureus isolates was detected, even after PBP2c induction (Table 1). Finally, all mecA-positive SNA isolates yielded positive results without PBP2a induction except for one Staphylococcus saprophyticus isolate, which tested positive only after induction (Table 1).
Therefore, the revised version of this immunochromatographic assay showed 100% sensitivity and specificity for detection of PBP2a from S. aureus but was unable to detect PBP2c. Regarding methicillin-resistant SNA isolates (n = 108), only 1 isolate was not directly detected (sensitivity of 99.1%). Cefoxitin induction increased the sensitivity to 100% for SNA isolates and did not induce false-positive results for methicillin-susceptible isolates. Of note, the red lines observed with methicillin-resistant SNA isolates were often less intense than those observed with MRSA isolates, indicating that the operator must strictly respect the inoculum recommendations of the manufacturer (one heaped 1-μl bacteriological loop of colonies) to avoid false-negative results. The high sensitivity for mecA-positive isolates is concordant with the results obtained with the previous version for S. aureus isolates (2–4, 9), and our study confirmed, using a large collection of species and isolates, that the performance of this revised assay was largely improved for SNA species, as reported by Canver et al. (6). Conversely, unlike with the previous version of the assay, no improvement in the detection of mecC-positive MRSA isolates was observed with cefoxitin induction, indicating that the antibodies and/or protocol used in the revised assay no longer allow the detection of PBP2c.
In conclusion, this study demonstrates that the revised version of the PBP2a SACCT is a highly reliable, easy-to-use, and rapid test to detect mecA-positive isolates, which are the most prevalent among methicillin-resistant staphylococcal clinical isolates. In contrast to the previous version, it can be used for rapid detection of methicillin resistance in SNA isolates in routine practice. However, users must be aware that this revised version no longer allows identification of mecC-positive MRSA isolates, even after induction. Thus, with this revised version of the test, for an S. aureus isolate phenotypically detected as being methicillin resistant using cefoxitin or oxacillin (with cefoxitin being more reliable for the detection of mecC-positive isolates [10]), mecC-positive MRSA can be suspected if the PBP2a SACCT result is negative. Conversely, in case of phenotypic doubt regarding methicillin resistance, such as with an atypical profile in automated methods (cefoxitin resistant and oxacillin susceptible), a negative PBP2a SACCT result, even after induction, does not allow exclusion of a mecC-positive isolate. Therefore, clinical microbiologists must keep in mind that isolates yielding negative results with this assay are not always oxacillin susceptible.
ACKNOWLEDGMENTS
We thank all of the laboratory technicians who participated in the study, as well as the French National Reference Centre for Staphylococci.
This work was supported by Alere, which provided all reagents for the present study but had no control or authority over the analysis, interpretation, and presentation of the results.
None of the authors has competing interests to declare.
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