Abstract
Using blaZ PCR as the “gold standard,” the sensitivities of CLSI penicillin zone edge and nitrocefin-based tests for β-lactamase production in Staphylococcus aureus were 64.5% and 35.5%, respectively, with specificity of 99.8% for both methods. In 2013, 13.5% of 3,083 S. aureus isolates from 31 U.S. centers were penicillin susceptible.
TEXT
Penicillinase-producing strains of Staphylococcus aureus emerged in the 1940s and by the 1970s represented 70 to 85% of the S. aureus population (1). Four types of blaZ genes (A to D) have been associated with penicillinase production in S. aureus (2). A study conducted in Germany demonstrated the sensitivity of nitrocefin-based testing was unacceptably low (36%) for penicillinase detection compared to blaZ PCR (3). Since 2012, the Clinical and Laboratory Standards Institute (CLSI) has recommended the penicillin zone edge test (4) to screen S. aureus isolates with a susceptible penicillin MIC (≤0.12 μg/ml) or disk zone (≥29 mm) for β-lactamase production (5).
There are limited data regarding the detection and prevalence of penicillin-susceptible S. aureus using a molecular method as the reference standard. The objectives of this national study were (i) to evaluate multiple phenotypic methods for β-lactamase detection in S. aureus using blaZ PCR as the “gold standard” and (ii) to determine the prevalence of penicillin-susceptible S. aureus in the United States.
As part of a national surveillance program, laboratories were asked to send 100 clinically significant S. aureus isolates to the University of Iowa. Isolates were recovered from specimens received during June to December 2013. Susceptibility testing using the CLSI broth microdilution method (5, 6) and mecA PCR were performed as previously described (7) on the 3,083 isolates received from 31 centers. The predominant specimen sources were 61% wound, 18% blood, 10% lower respiratory tract, 4% tissue, and 2% sterile body fluid. Classification as methicillin-susceptible S. aureus (MSSA) was based on a negative mecA PCR result. All isolates with a susceptible penicillin MIC (≤0.12 μg/ml) were assessed for β-lactamase production using blaZ PCR, penicillin zone edge, and induced nitrocefin-based testing. PCR to amplify a 355-bp region of the blaZ gene was followed by sequencing to classify positive strains as type A, B, C, or D as previously described (8). The blaZ type was also determined for a subset of penicillin-resistant isolates (n = 51). The penicillin zone edge test was performed on Mueller-Hinton agar using a 10-U penicillin disk following CLSI guidelines (5). After 16 to 18 h of incubation in ambient air, a sharp zone edge was interpreted as positive and a fuzzy zone as negative for β-lactamase production. Nitrocefin-based testing was performed on induced growth taken from the zone margin surrounding a 10-U penicillin disk.
As expected, all 1,387 mecA-positive strains had penicillin MICs in the resistant range (≥0.5 μg/ml; 80% at >16 μg/ml). There were 448 isolates (14.5% of all isolates, 26.4% of MSSA isolates) with a susceptible penicillin MIC of ≤0.12 μg/ml. Thirty-one of the 448 isolates (6.9%) were blaZ positive (Table 1) and represented 32.1% of 53 isolates with a penicillin MIC of 0.12 μg/ml and 3.8% of 370 isolates with a penicillin MIC of 0.06 μg/ml. None of the 25 isolates with a penicillin MIC of ≤0.03 μg/ml contained blaZ.
TABLE 1.
blaZ PCR results for 448 MSSA isolates with susceptible penicillin MICsa
| Penicillin MIC (μg/ml) | No. of isolates | No. (%) of isolates: |
|
|---|---|---|---|
| blaZ positive | blaZ negative | ||
| ≤0.015 | 1 | 0 (0) | 1 (100) |
| 0.03 | 24 | 0 (0) | 24 (100) |
| 0.06 | 370 | 14 (3.8) | 356 (96.2) |
| 0.12 | 53 | 17 (32.1) | 36 (67.9) |
| Total | 448 | 31 (6.9) | 417 (93.1) |
Susceptibility was defined as a MIC of ≤0.12 μg/ml.
Phenotypic β-lactamase screening test results for the 31 blaZ-positive isolates are shown in Table 2. One blaZ PCR-negative isolate (penicillin MIC, 0.12 μg/ml) was positive by both phenotypic tests. The sensitivity and specificity of penicillin zone edge testing were 64.5% and 99.8%, respectively. The sensitivity and specificity of the induced nitrocefin testing were 35.5% and 99.8%, respectively. These findings are similar to those from a German study that included 197 S. aureus isolates with Vitek 2 penicillin-susceptible results and reported sensitivities of 71.4% for penicillin zone edge and 35.7% for nitrocefin testing with blaZ PCR as the reference standard (3). A small Australian study assessed 50 S. aureus isolates that appeared penicillin susceptible by disk diffusion but only found 2 blaZ-positive isolates for evaluation of phenotypic methods (with one isolate detected by penicillin zone edge and neither detected by nitrocefin testing) (9). A larger Australian evaluation analyzing 157 isolates that appeared penicillin susceptible by agar dilution (38 were blaZ positive) noted 89% sensitivity and 100% specificity for the CLSI zone edge test (10). The EUCAST zone edge test incorporating a lower-concentration (1-U) penicillin disk provided 100% sensitivity and specificity for penicillinase detection in the Australian study (10). Lack of a CLSI quality control range and limited availability of the 1-U penicillin disk hinder further investigation of the EUCAST zone edge test. A U.S. study evaluating 105 isolates that appeared penicillin susceptible by disk diffusion found 10 (9.5%) to possess blaZ and noted variability among four readers of the CLSI penicillin zone edge test (with only 60% of those 10 strains having a sharp edge reported by all readers) (11).
TABLE 2.
Phenotypic test results for 31 blaZ-positive isolates with susceptible penicillin MICs
| Penicillin MIC (μg/ml) | No. of isolates | No. (%) of isolates detected by: |
|
|---|---|---|---|
| Penicillin zone edge | Inducible nitrocefin test | ||
| 0.06 | 14 | 4 (28.6) | 3 (21.4) |
| 0.12 | 17 | 16 (94.1) | 8 (47.1) |
| Total | 31 | 20 (64.5) | 11 (35.5) |
The distribution of β-lactamase types detected in the present study among blaZ-positive isolates with a penicillin MIC of ≤0.12 μg/ml was 26% A, 13% B, and 52% C. (Three isolates did not correspond to a known type [NT].) The distribution was similar for isolates with higher penicillin MICs (Table 3). A 2009 study analyzing 98 MSSA isolates reported no blaZ for 13%, type A β-lactamase for 26%, type B for 15%, and type C for 46% (8). A South Korean study found type A blaZ in 17%, type B in 20%, type C in 53%, and type D in 1% of 220 MSSA isolates (12). Cefazolin failures have been reported for MSSA infections caused by type A blaZ isolate demonstrating an inoculum effect (8), but other retrospective studies have only found treatment success (12). Prospective studies are needed to determine if screening MSSA isolates for a cefazolin inoculum effect has clinical utility.
TABLE 3.
blaZ types detected in S. aureus isolates with susceptible and resistant penicillin MICs
| Penicillin MIC (μg/ml) | No. (%) of isolates: |
No. of isolates with blaZ type detected |
||||
|---|---|---|---|---|---|---|
| Tested for blaZ | blaZ positive | A | B | C | NT | |
| Susceptibility | ||||||
| 0.015 | 1 (100) | |||||
| 0.03 | 24 (100) | |||||
| 0.06 | 370 (100) | 14 (3.4)a | 2 | 2 | 8 | 2 |
| 0.12 | 53 (100) | 17 (32.1)b | 6 | 2 | 8 | 1 |
| Resistance | ||||||
| 0.25 | 8 (14.8) | 8 (100) | 5 | 3 | ||
| 0.5 | 1 (0.7) | 1 (100) | 1 | |||
| 1 | 10 (3.0) | 10 (100) | 3 | 3 | 4 | |
| 2 | 4 (4.5) | 4 (100) | 2 | 1 | 1 | |
| 4 | 10 (3.8) | 10 (100) | 2 | 8 | ||
| 8 | 11 (2.6) | 11 (100) | 3 | 1 | 7 | |
| 16 | 3 (1.6) | 3 (100) | 1 | 1 | 1 | |
| >16 | 4 (0.4) | 4 (100) | 1 | 3 | ||
| Total | 499 | 82 | 25 | 10 | 43 | 4 |
The blaZ types of the 10 isolates with a penicillin MIC of 0.06 μg/ml and not detected by the penicillin zone edge test were types A (n = 2), B (n = 1), C (n = 6), and nontypeable (NT [n = 1]).
The blaZ type of the isolate with a penicillin MIC of 0.12 μg/ml and not detected by the penicillin zone edge test was type A.
A correlation of penicillin zone edge test performance with blaZ type was not apparent in the present study. The 11 isolates not detected by the zone edge method represented all of the blaZ types (37% of the 8 type A, 25% of the 4 type B, 38% of the 16 type C, and 33% of the 3 nontypeable [NT] isolates).
In conclusion, the CLSI penicillin zone edge method detected penicillinase production among 45% more blaZ-positive S. aureus isolates than nitrocefin-based testing. The failure of the CLSI zone edge test to detect 35% of blaZ-positive isolates is concerning. This study indicates that in the United States, 13.5% of S. aureus isolates (24.6% of MSSA isolates) are penicillin susceptible based on negative blaZ testing. Many labs do not routinely test for penicillin susceptibility (13, 14). Although higher mortality has been reported for cefuroxime therapy of penicillin-susceptible MSSA bacteremia (15), superiority of penicillin over penicillinase-stable agents (e.g., dicloxacillin, nafcillin, and oxacillin) has not been proven. The limitations of phenotypic testing should be relayed to clinicians who request assessment of penicillin susceptibility. Molecular testing for blaZ is recommended before relying on penicillin for therapy of complicated MSSA infections.
(This work was presented in part at the 54th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 7 September 2014 [16].)
ACKNOWLEDGMENTS
We thank the participating medical centers for providing the isolates of S. aureus characterized in this study.
S.S.R. has received research funding from bioMérieux, BD Diagnostics, OpGen, Forest Laboratories, Nanosphere, and Pocared. D.J.D. has received research funding from Merck, Pfizer, Schering-Plough, Astellas, and bioMérieux. G.V.D. has received research funding from Abbott Laboratories, Schering-Plough, Bayer Pharmaceutical, Merck, Shionogi, Cubist, and Astra-Zeneca. He has been on the speakers' bureaus of Abbott Laboratories, Aventis, Astra-Zeneca, Forest Laboratories, Pfizer, Astellas, and Schering-Plough. All other authors report no conflicts of interest.
Financial support for this project was provided by Allergan Laboratories, Inc. (New York, NY).
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