Rapid detection of methicillin-resistant Staphylococci by multiplex PCR

Journal of Hospital Infection (1999) 43: 33–37

Rapid detection of methicillin-resistant Staphylococci by multiplex PCR A. M. Kearns, P. R. Seiders, J.Wheeler, R. Freeman and M. Steward* Public Health Laboratory, General Hospital,Westgate Road, *Novocastra Laboratories, Balliol Business Park West, Benton Lane, Newcastle upon Tyne, UK Summary: A multiplex PCR was developed to detect the coagulase gene (coa; pathognomic of Staphylococcus aureus) and the mecA gene (characteristically encoding for methicillin resistance in staphylococci) in a single, rapid test. Suitable primers for the gene targets and an internal, amplification control were incorporated into a multiplex PCR assay, which was then optimized on a capillary air thermal cycler to improve the turnaround time of the test to approximately 1·5 hours. The assay was evaluated with 111 fresh clinical isolates of staphylococci. The multiplex PCR correctly distinguished between isolates of S. aureus, which were sensitive to methicillin (MSSA) and those resistant to it (MRSA). It also correctly differentiated between similar isolates of coagulase negative staphylococci (MSSE and MRSE respectively). It was concluded that this multiplex PCR was a rapid and reliable method for the detection of methicillin-resistant staphylococci. © 1999 The Hospital Infection Society

Key words: The Multiplex PCR; staphylococci; Staphylococcus aureus; methicillin-resistant; MRSA; detection.

Introduction Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA), and distinction from other methicillin-resistant staphylococci (MRSE), has become imperative in view of the increasing incidence of nosocomial infections caused by these pathogens.1,2 Commercially available kits, based on agglutination reactions, do not always provide clear-cut results.3 The coagulase status of an isolate is not always easily established in a timely fashion, incurring delays in the definitive identification of S. aureus strains. Susceptibility testing is difficult since it is dependent on the rigorous control of assay Received 7 August 1998; revised manuscript accepted 5 May 1999. Address correspondence to: Prof. R. Freeman, Public Health Laboratory, General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE, England.

0195–6701/99/090033 + 5 $12.00

conditions.4–6 It usually entails a delay of at least 24 h after primary isolation. Even under optimal conditions, some staphylococcal isolates exhibit intermediate levels of methicillin susceptibility in vitro, which are hard to interpret in terms of clinical management. In this study, the use of PCR to detect the coagulase gene (coa; pathognomic for S. aureus) and the mecA gene (characteristically encoding for methicillin resistance in staphylococci) in a single rapid test, with an internal amplification control, was investigated.

Materials and methods Bacterial isolates and culture conditions

One hundred and eleven fresh clinical isolates of staphylococci were examined (Table I). © 1999 The Hospital Infection Society

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Table 1 Multiplex PCR results obtained with 111 clinical isolates of staphylococci Identity

No. of isolates with: mecA coa

No. of isolates examined

MSSA* MRSA† MSSE‡ MRSE§

0 31 0 28

30 31 22 28

30 31 0 0

*MSSA, Methicillin-sensitive S. aureus; †MRSA, Methicillin-resistant S. aureus; ‡MSSE, Methicillin-sensitive, coagulase negative staphylococci; §MRSE, Methicillin-resistant, coagulase negative staphylococci

These included 30 isolates of methicillin-susceptible S. aureus and 31 of methicillin-resistant S. aureus (MRSA). Fifty isolates of coagulase negative staphylococci, of which 22 were susceptible to methicillin (MSSE) and 28 methicillin-resistant (MRSE), were also tested. The isolates were cultured on Columbia blood agar (Oxoid Ltd., Basingstoke) or DST agar (Oxoid Ltd.) overnight prior to DNA extraction and PCR testing. Coagulase detection and susceptibility testing

Conventional slide coagulase testing with human plasma and standard disc diffusion susceptibility testing were used to establish the coagulase and methicillin-susceptibility status of all the isolates studied. DNA purification from culture samples

The original protocol for DNA extraction was adapted from that described by Ünal et al.7 as follows. An aliquot of an overnight culture (approximately 108 cells) was suspended in 50 µl of lysostaphin (100 µg/ml; Sigma-Aldrich Company Ltd., Poole, Dorset). After incubation at 37°C for 10 min, 50 µl proteinase K (100 µg/ml; Sigma-Aldrich Company Ltd.) and 150 µl 100 mM Tris pH7·5 were added. The suspension was incubated at 37°C for a further 10 min, boiled for 5 min and centrifuged for 2 min. One microlitre of the supernatant, containing the extracted bacterial DNA, was used

in the PCR assay. Aliquots of extracted DNA were stored at –20°C prior to use. PCR amplification

PCR amplification was performed using a S. aureus primer set (NCL-SA-PS; Novocastra Laboratories, Newcastle upon Tyne, UK). Briefly, this consisted of two pairs of primers. Primers SA-1 (5′ CGG TAA CAT TGA TCG CAA CGT TCA 3′) and SA-2 (5′ CTT TGG AAC GAT GCC TAA TCT CAT 3′) amplified a 214-bp fragment of the mecA gene. Primers SA-3 (5′ GTA GAT TGG GCA ATT ACA TTT TGG AGG 3′) and SA-4 (5′ CGC ATC AGC TTT GTT ATC CCA TGT A 3′) amplified a 117-bp fragment from the coa gene. In addition, the set included an internal control template. This co-amplified with the mecA primers to yield a 150 bp product, which served as an amplification control and aided in the detection of false negative results. One microlitre of DNA sample was added to 49 µl of PCR mixture consisting of 4 mM MgCl2, 200 µM dNTPs, 15 pmol each primer, 25 fg internal control and 1 U Taq DNA polymerase. The assay was initially performed according to the manufacturers’ instructions, using a solid-block thermal cycler, with only minor modifications to the cycling profile, as described below. After an initial denaturation step (1 min at 94°C), 15 cycles of amplification were performed as follows: denaturation at 94°C for 30 s, annealing at 68°C for 30 s, and DNA extension at 72°C for 30 s. Thereafter, a further 20 cycles of amplification were completed as above, but using an annealing temperature of 60°C. The reaction was achieved with a final extension at 72°C for 2 min. Amplification was carried out on a GeneAmp PCR System 2400 (Perkin Elmer, Warrington, Cheshire); the cycle running time was 1·5 h. Amplified products were separated by electrophoresis at 110V for 30 min through a 3% agarose gel and visualized using a transilluminator. The resulting banding profiles were examined for the presence/absence of target bands. After initial evaluation, the PCR assay was optimised on a capillary air thermal cycler

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Figure 1 An ethidium bromide-stained gel demonstrating the typical banding patterns observed with the multiplex PCR assay. Lane 1: methicillinsensitive S. aureus; lane 2: methicillin-resistant S. aureus; lane 3: methicillin-resistant coagulase negative staphylococci; lane 4: methicillin-sensitive coagulase negative staphylococci; lane 5: 50-bp molecular weight ladder.

(CATC; Rapidcycler, BioGene Ltd., Kimbolton, Cambridgeshire) to capitalise on its greater efficiency.8 The mastermix comprised 4 mM MgCl2/BSA/Sucrose/Dye (BioGene Ltd.), 200 µM dNTPs, 15 pmol each primer, 25 fg internal control and 0·25 U Taq DNA polymerase. The reaction mixture consisted of 1 µl DNA extract and 9 µl mastermix. The cycling profile comprised an initial step of 94°C for 1 min, followed by 15 cycles of 94°C for 0 s, 65°C for 0 s, 72°C for 4 s, then 25 cycles of 94°C for 0 s, 60°C for 0 s, 72°C for 4 s, and finally 72°C for 30 s. The amplified products were electrophoresed and visualized as described above. The cycling time using the CATC was reduced to <20 minutes and the entire assay, including DNA extraction, amplification and detection took approximately 1·5 hours.

Results An ethidium bromide-stained gel demonstrating the typical banding patterns observed with

MRSA, MSSA, MRSE and MSSE is shown in Figure 1. The amplicon sizes of the mecA, internal control and coa were 214-, 150- and 117-bp respectively and, thus, were clearly distinguishable on an agarose gel. The multiplex PCR results obtained with 111 isolates of staphylococci are summarised in Table I. All gave unambiguous banding profiles.

Discussion Both MRSA and MRSE are significant pathogens in community-acquired and nosocomial infections.1,2 Methicillin resistance in S. aureus and coagulase-negative species is primarily mediated by the overproduction of PBP 2a. This altered penicillin-binding protein has extremely low affinities for β-lactam antibiotics.9 The structural determinant encoding PBP2a is the mecA gene, which has high levels of homology in MRSA and MRSE10 but is absent from susceptible staphylococcal

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isolates.11,12 The femA-femB operon is a chromosomally-determined factor that acts as a regulator gene, essential for the expression of methicillin resistance in S. aureus.13 Detection of femA, which appears to be unique to S. aureus, may be used to identify the species.7 Previous studies have evaluated the feasibility of using PCR methodology for the detection of methicillin resistance and for the identification of S. aureus strains on the basis of mecA14 and femA7 gene amplification, respectively, or both, in a multiplex assay.15 Others have included an internal control, such as the 16S rRNA gene target, to identify potential falsenegative results.16 Good correlation was reported between the detection of mecA and methicillin-resistance but the assays described took 6 to 8 h to complete. This limits their usefulness in a routine diagnostic laboratory. More recently, a multiplex PCR detecting staphylococcal mecA and coa genes has been described17, with an assay time of only 4 h. The method involved emulsifying colonial material directly into the PCR amplification reaction mixture i.e., there was no extraction of bacterial DNA prior to amplification. Initial investigations in this laboratory failed to reproduce those results. Hence, it was decided to develop an assay using a more traditional approach for sample preparation. All the above assays have used solid-block thermal cyclers and PCR reaction mixtures of at least 50 µl. Thus, they have taken several hours to complete and are relatively expensive in consumable costs. These are prime concerns for a routine diagnostic laboratory, which might wish to adopt such techniques as a screening tool, with a high throughput of isolates. The multiplex PCR assay described herein proved to be simple to perform, very rapid and robust. All the clinical isolates were correctly characterized by it as judged by conventional coagulase and methicillin susceptibility testing results. The PCR results were sometimes more clear-cut and easier to interpret than those of the other tests. The internal control was a useful amplification/inhibition control. Its inclusion in the assay did not interfere with the detection of either of the targets and enabled

A. M. Kearns et al.

negative results to be interpreted with confidence. The total assay time, including extraction of staphylococcal DNA, was only about 1·5 hours when using a CATC. Thus, the test could be used in a situation where conclusive identity of a staphylococcal isolate is needed urgently. A further advantage of using CATC technology is the small volume of the PCR reaction mixture employed (10 µl as opposed to 50 µl). This reduces the consumable cost of the assay by a factor of 5; as performed on the CATC, it costs ≤£1·50 per test. A study is underway to evaluate an abbreviated DNA extraction procedure, using only the lysostaphin step prior to boiling, which would simplify this process and further reduce the assay’s turnaround time. The widespread adoption of this rapid and simple assay has the potential to improve individual patient management and both community-acquired and nosocomial infection control procedures.

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