Real-Time PCR Assay for Detection of blaZ Genes in Staphylococcus aureus Clinical Isolates

Abstract

The Clinical and Laboratory Standards Institute recommends consideration of blaZ gene testing for cases of serious Staphylococcus aureus infection. Conventional PCR methods have demonstrated superior sensitivity and specificity to phenotypic tests. To our knowledge, this is the first description of real-time PCR detection of the blaZ gene.

TEXT

Staphylococcus aureus bacteremia is a cause of significant morbidity and mortality, with 30-day mortality rates of up to 27% (1). Although rates of S. aureus penicillin susceptibility are generally low, ranging from 5 to 20% (2, 3), penicillin remains the treatment of choice for penicillin-susceptible S. aureus (PSSA) based on in vitro data indicating increased susceptibility to penicillin over penicillinase-resistant β-lactams (3–5).

Penicillin resistance in S. aureus is manifest predominantly via the production of β-lactamase encoded by the blaZ gene (6), with four variants of the β-lactamase (A, B, C, and D) identified using immunologic methods and substrate profile techniques (7, 8). A number of phenotypic methods for the detection of β-lactamase production in Staphylococcus species have been investigated (9–15), but compared to detection of the blaZ gene by PCR, all phenotypic methods had a sensitivity of less than 72% (15, 16). This has led to the Clinical and Laboratory Standards Institute (CLSI) recommending that blaZ gene detection be considered for PSSA isolates from cases of serious infection requiring penicillin therapy (4).

Various PCR methods have been described for the detection of blaZ gene in Staphylococcus species (5, 16), but to date there are no published real-time PCR methods. We describe a real-time PCR assay for the detection of the blaZ gene in S. aureus.

One hundred nonduplicate S. aureus bloodstream isolates (BSI) collected between September 2011 and December 2012 by the PathWest Laboratory Medicine WA Queen Elizabeth II Medical Centre Department of Microbiology were included in this study. Of these, 50 isolates were susceptible to penicillin and 50 were resistant to penicillin when tested by Kirby-Bauer disc diffusion according to CLSI guidelines (17). An additional 28 nonduplicate clinical S. aureus isolates which had the presence or absence of the blaZ gene determined by microarray analysis (Alere Technologies, Jena, Germany), as previously described (18), were also included. Of these, 14 isolates had blaZ detected by microarray, and 14 isolates did not. Previously characterized (8, 19–22) reference S. aureus type strains and β-lactamase strain variants were used as positive controls. These included the following: type A, PC1(pI254) and NCTC 9789; type B, 22260 and ST79/741; type C, 3804(pII3804), RN9(pII147), and V137; and type D, FAR10.

Colonies of S. aureus located at the penicillin zone edge of a 10-μg penicillin disc on Mueller-Hinton agar (Oxoid, Ltd., Hampshire, United Kingdom) were tested with nitrocefin-impregnated discs (BBL; BD, USA) according to the manufacturer's instructions. Zone edge assessment was made on all isolates and recorded as being either a sharp “cliff” edge suggestive of β-lactamase production or a tapered “beach” edge suggestive of absence of β-lactamase production. Interpretation of phenotypic tests was performed by two independent observers without reference to the PCR results. Although interobserver variability has been demonstrated previously in regard to interpretation of zone edge characteristics (15), in this study no interobserver discrepancies were demonstrated.

Bacterial DNA from S. aureus isolates cultured on horse blood agar for 24 h at 37°C in room air was extracted as follows. Two to five loopfuls of S. aureus were suspended in 1 ml sterile demineralized water and boiled for 10 min, and the suspension was centrifuged for 2 min. From this, 8 μl of the supernatant was used with 12 μl of either conventional or real-time PCR amplification mixes.

The conventional blaZ PCR was performed as described previously (16). The real-time blaZ PCR primer and probe sequences were designed in-house using Primer Express software (Applied Biosystems). Fluorophore-labeled oligonucleotide probes were synthesized by Applied Biosystems (MGB probes), and primers were synthesized by Integrated DNA Technologies (Fisher Biotec, Australia). Sequences for the blaZ gene available from GenBank were obtained by BLASTn search using accession no. FR714929 and aligned prior to primer and probe design. Anomalous bases were used in the primers and probe where necessary to allow detection of genetic variants of the blaZ target. The reaction mix contained PCR buffer (Applied Biosystems, USA), 4 mM MgCl₂, 0.2 mM deoxynucleoside triphosphates (Sigma, USA), 0.75u AmpliTaq Gold DNA polymerase (Applied Biosystems, USA), 0.2 μM forward primer (5′-GCTTTAAAAGAACTTATTGAGGCTTCA-3′), 0.2 μM reverse primer (5′-CCACCGATYTCKTTTATAATTT-3′), and 0.2 μM TaqMan probe (5′-FAM-AGTGATAATACAGCAAACAA-MGBNFQ-3′, where FAM is 6-carboxyfluorescein). The amplification was performed using RotorgeneQ real-time thermocyclers (Qiagen, Australia). Reactions were run under the following conditions: 95°C for 10 min, followed by 50 cycles of 12 s at 94°C, 15 s at 55°C, and 20 s at 72°C. Probe emission signals were acquired during the 20-s extension step of the cycling program.

Amplified DNA produced by the conventional and real-time PCR was sequenced using the AB BigDye Terminator version 3.1 sequencing kit on the AB 3130xl genetic analyzer (Applied Biosystems). Sequences were compared to those of the blaZ gene available in GenBank using BLASTn software.

The conventional PCR method was verified by testing with the positive-control reference strains and the clinical isolates previously characterized by microarray analysis. All positive-control reference strains had blaZ detected by both conventional PCR and real-time PCR. The 14 clinical isolates that had blaZ detected by microarray analysis, demonstrating cliff penicillin zone edges, were nitrocefin test positive and had blaZ detected by both conventional and real-time PCR. The 14 clinical isolates that did not have blaZ detected by microarray analysis, demonstrating beach penicillin zone edges, were nitrocefin test negative and did not have amplified DNA produced by either conventional or real-time PCR.

The 50 S. aureus BSI that tested penicillin resistant by disc diffusion according to CLSI criteria had blaZ detected by both the conventional PCR and real-time PCR. For these isolates, all had either no zone around the penicillin disc or a cliff zone edge, and all had a positive nitrocefin test; results are shown in Table 1. For the 50 phenotypically penicillin-susceptible S. aureus BSI, the sensitivity, specificity, and positive and negative predictive values of the real-time PCR, the nitrocefin disc test, and the characterization of zone edge, compared to those of the conventional PCR reference method, are shown in Table 1. Two of these had blaZ detected by both the conventional and real-time PCR methods, and the results were confirmed by sequencing. Sequences of the 2 isolates demonstrated 100% homology with blaZ types in GenBank (accession no. DQ016067 and GQ980074). There was no genetic mutations identified that would account for the discrepant phenotypic and genotypic results; however, expression of the blaZ gene may be affected by mutations in DNA encoding promoter or repressor regions (23). These 2 isolates had a negative nitrocefin test, and one of the isolates had a penicillin zone edge characterized as beach edge and the other a cliff edge. All of the remaining 48 BSI that did not have blaZ detected by PCR had phenotypic test results consistent with the absence of β-lactamase.

TABLE 1.

Phenotypic and real-time PCR tests compared to conventional PCR for detection of β-lactamase in S. aureus isolates

Testing method and result by S. aureus isolate group	No. of positive tests	No. of negative tests	% sensitivity	% specificity	PPV (%)a	NPV (%)b
Penicillin-sensitive (by disc diffusion) S. aureus BSI (n = 50)
Nitrocefin disc test	0	50	0	100	0	96
Penicillin zone edgec	1	49	50	100	100	98
Real-time PCR	2	48	100	100	100	100
Conventional PCR	2	48
Penicillin-resistant (by disc diffusion) S. aureus BSI (n = 50)
Nitrocefin disc test	50	0	100		100
Penicillin zone edgec	50	0	100		100
Real-time PCR	50	0	100		100
Conventional PCR	50	0
S. aureus clinical isolates, blaZ status characterized by microarray (n = 28)
Nitrocefin disc test	14	14	100	100	100	100
Penicillin zone edgec	14	14	100	100	100	100
Real-time PCR	14	14	100	100	100	100
Conventional PCR	14	14
S. aureus blaZ-positive reference strains (n = 6)
Nitrocefin disc test	6	0	100		100
Penicillin zone edge	6	0	100		100
Real-time PCR	6	0	100		100
Conventional PCR	6	0
All S. aureus isolates tested (n = 134)
Nitrocefin disc test	70	64	97.2	100	100	96.9
Penicillin zone edgec	71	63	98.6	100	100	98.4
Real-time PCR	72	62	100	100	100	100
Conventional PCR	72	62

^aPPV, positive predictive value.

^bNPV, negative predictive value.

^cA beach edge indicates lack of β-lactamase (negative test), whereas a cliff edge indicates its presence (positive test).

The real-time PCR for detection of the blaZ gene was developed in response to current CLSI recommendations. Conventional PCRs have previously been described and have been used as the gold standard for β-lactamase detection when comparing phenotypic tests (6, 13, 16, 24, 25). The advantages of the real-time PCR over conventional PCR include a faster turnaround time, less specimen handling with subsequent reduced workload and risk of specimen contamination, lower cost, and equivalent sensitivity (100%) and specificity (100%). These advantages would facilitate more routine testing of the blaZ gene in accordance with CLSI recommendations. The clinical impact of introducing routine real-time PCR testing will require further study.

To our knowledge, this is the first published study to describe a real-time PCR for the detection of the blaZ gene in S. aureus. This real-time PCR is able to detect all four variants of S. aureus β-lactamase facilitating rapid and accurate confirmation of the presence of the blaZ gene in S. aureus clinical isolates that test penicillin susceptibility by phenotypic methods.