A Novel Staphylococcal Cassette Chromosome mec Type XI Primer for Detection of mecC-Harboring Methicillin-Resistant Staphylococcus aureus Directly from Screening Specimens

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) screening using real-time PCR has decreased in sensitivity due to the emergence of variant staphylococcal cassette chromosome mec element (SCCmec) types. We have designed and validated a novel SCCmec XI primer, which enables for the first time the rapid detection of mecC-harboring MRSA directly from nasopharyngeal swabs without prior cultivation.

TEXT

Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health problem affecting the therapy of nosocomial, community-acquired, and livestock-associated infections and requiring the implementation of infection control and preventive measures (1). Resistance to β-lactam antibiotics is mediated by expression of penicillin-binding protein PBP2a. The encoding gene, mecA, is localized on the mobile staphylococcal cassette chromosome mec element (SCCmec), which exists in variant types and subtypes (http://www.sccmec.org). A rapid screening for MRSA-colonized patients is possible using a SCCmec-orfX-based MRSA-screening PCR. The initial TaqMan PCR described in 2004 by Huletsky et al., comprised five SCCmec forward primers targeting the right extremity of the main SCCmec types and a reverse primer targeting the S. aureus-specific orfX gene (2), thus amplifying an entity within the MRSA genome sequence, which was detected by a degenerate TaqMan probe. A modified PCR published in 2011 extended the MRSA screening to rare SCCmec types (3).

In 2011, a highly divergent mec gene, mecC, on novel SCCmec element XI, was discovered in MRSA strains of domestic and wildlife animals (4–6) and of humans (7–9).

Recently, MRSA screening cultures of two patients in Düsseldorf revealed two MRSA isolates, Sc16837 and Sc26644, with unusual resistance patterns (oxacillin susceptible, cefoxitin screening positive), arousing the suspicion of mecC-positive MRSA strains (10). Both strains were susceptible to all tested antibiotics other than β-lactams, as described for mecC-positive MRSA strains from human and animals in Europe (11–15). An in-house MRSA multiplex PCR detected the S. aureus-specific nuc gene but did not detect the mecA nor the PVL gene (Tables 1 and 2) (16). The spa typing revealed spa types t843 and t1736. Type t843 has already been associated with mecC-positive MRSA strains in humans and cattle in northern Europe (6, 17). Presence of the mecC gene was verified with mecC real-time PCR (6) with subsequent sequencing and BLAST analysis of the 357-bp PCR products. The embedded regions of the primers showed 100% sequence homology to respective mecC gene regions of S. aureus LGA251 (GenBank accession no. FR821779) and other SCCmec type XI strains (e.g., GenBank accession nos. LK024544, HF569116, and JN794592).

TABLE 1.

Primers and probes of MRSA-screening real-time PCR

Primer/probe	Oligonucleotide sequence, 5′ to 3′	μM per PCR	SCCmec typea	Reference
F1/IC-Fb	GTCAAAAATCATGAACCTCATTACTTATG	0.2	II, IV, IVa, VI, VIII	2
F2	AATATTTCATATATGTAATTCCTCCACATCTC	0.2	II, IIa, V, VII	3
F3	CTTCAAATATTATCTCGTAATTTACCTTGTTC	0.2	III	3
F4	CTCTGCTTTATATTATAAAATTACGGCTG	0.2	IVe	2
F5	TCACTTTTTATTCTTCAAAGATTTGAGC	0.2	II, IVa	3
F10	CCGCTCCTTTTATATTATACACAACCTATT	0.2		3
F11	GCCATATTAATGCCTCACGAAAC	0.2	I, II, IV, X	3
F13	TCCCTTTATGAAGCGGCTGAA	0.2	I, II, IV, IVa, IVc, IVq, IX	3
F14	AAGCTATAGTTCAGCATTATCGTAAGTTAACT	0.2	IV, IVa	3
F17	ACTCTGATAAGCCATTCATTCATCCA	0.2		3
F18	ACAATCCTAACATAAGATTGTGGCTTT	0.2		3
F20	GCATATTCACTTTGATAAGCCATTCAT	0.2	IVk	3
F24	CCCAAACTCTTAACTTTCTTCAATACATT	0.2		3
F25	TTCTAAGGTAGCTTCCCTTTCAATAATTT	0.2	V	3
R1	CGTCATTGGCGGATCAAAC	0.2		3
R2	CGTCATTGGTGGATCAAACG	0.2		3
Probe	FAM-CACAARGAYGTCTTACAACG-MGBNFQ	0.1		3
IC-R	GGATCAAACGGCCTGCACA	0.2		2
IC probe	HEX-ATGCCTCTTCACATTGCTCCACCTTTCCT-BHQ1	0.02		Adapted to 2
(+/−)
SCCmec XI	GATAACTCTCGCAAAACATAACG	0.2	XI	This study

^aDefined by BLAST analysis.

^bF, forward primer; R, reverse primer; (+/−), addition of SCCmec XI primer, as indicated.

TABLE 2.

MRSA isolates of SCCmec type XI in MRSA-screening and MRSA-multiplex PCRs

MRSA strain	SCCmec type	spa type	Original MRSA screening (CTa)	MRSA screening + SCCmec XI (CT)	nuc (CT)	mecA (CT)	mecC (CT)
07-03165	I	t001	22	21	21	19
10-01267	I	t041	22	22	22	20
12-00241	I	t001	19	18	19	16
08-00463	II	t003	20	21	21	18
14-00535	II	t003	21	20	22	19
12-02225	II	t003	21	20	21	18
01-00694	III	t030	17	17	13	15
12-01196	III	t030	21	20	16	18
12-00444	III	t037	21	22	15	17
09-01691	IVa	t008	18	19	15	17
09-02003	IVa	t008	20	19	15	17
09-00417	IVa	t008	19	19	15	17
04-01872	IVc	t019	20	21	17	18
03-02782	IVc	t008	20	20	16	19
04-01389	IVd	t036	19	18	15	17
12-02344	V	t011	20	21	16	18
12-03117	V	t3091	20	20	16	18
14-03203	V	t011	19	20	15	17
13-03522	V	t7656	20	20	20	17
06-01359	VI	t640	21	21	21	19
06-00468	VI	t311	22	21	21	18
EQC	XI	t10009		20	24		25
Sc16837	XI	t1736		23	21		20
Sc26644	XI	t843		25	21		22
DD17b	XI	t843		26	19		17
SVA15b	XI	t843		27	21		24
DD158b	XI	t843		26	20		23
Z4070b	XI	t843		24	18		17
Z4183b	XI	t843		26	19		18
S130-13b	XI	t843		25	19		18
DD44b	XI	t5771		25	19		18

^aC_T, cycle threshold.

^bMRSA strains according to Schlotter et al. (5).

To date, the detection of MRSA with the SCCmec type XI element is rare in humans. Nevertheless, its occurrence might have been underestimated, as it depends on strain cultivation. A rapid molecular screening approach by the commonly used SCCmec-orfX-based MRSA-screening real-time PCR has been hindered by the high variability of the SCCmec type XI element (2, 3). Although the mecC-carrying MRSA strains Sc26644 and Sc16837 presented here derived from routine screening of asymptomatic patients, other clinical mecC-derived MRSA isolates that cause severe soft tissue infections, osteomyelitis, and bacteremia have been described (8, 18, 19). Thus, rapid detection of mecC-derived MRSA is necessary, and methods have recently been developed to resolve this deficiency. Commercial systems have been upgraded by the addition of mecC detection. Becker et al., published in 2013 a modified version of a commercial, modular multiplex-PCR MRSA detection assay, in which labeled oligonucleotides were used for the detection of nuc as the S. aureus species-specific gene, mecA, and mecC (20). The group of Nijhuis et al., developed a high-throughput screening approach by the use of combined real-time PCR assays which were based on overnight selective broth enrichment (21).

All of these methods require isolation of the strain prior to testing, as they have a common potential source of error: The simultaneously amplified S. aureus and mecC gene regions may not necessarily be located on the same genome but may be derived from a mixture of oxacillin-susceptible S. aureus (MSSA) and mecC-positive coagulase-negative staphylococcus (CoNS) if the test is performed directly from swab sample.

At the University Clinic of Düsseldorf, the MRSA-screening real-time PCR was customized with respect to locally occurring MRSA variants. This TaqMan PCR was carried out on a CFX96 real-time PCR machine (Bio-Rad, Munich, Germany) in a total volume of 25 μl containing 2.5 μl DNA lysate of a swab specimen in NoROX PCR mastermix (Qiagen, Hilden, Germany) with primers and probes concentrations given in Table 1. Thermal cycling conditions were as follows: 10 min at 95°C for initial denaturation followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. Fifty copies of an internal control (IC) plasmid were added as an inhibition control prior to extraction, consisting of an artificial 128-bp insert with the IC-R primer binding site (bp 1 to 19), a genomic sequence of Drosophila melanogaster as the target of the IC probe (bp 49 to 77), and the F1-primer binding site (bp 100 to 128).

Based on an alignment of the 2.25-kb SCCmec regions of mecC- and mecA-carrying MRSA strains, which comprised the regions from the binding site of the mecC forward primer to that of the orfX reverse primer, we designed a novel SCCmec primer, SCCmec XI (5′-GATAACTCTCGCAAAACATAACG-3′) which targeted all published SCCmec type XI right extremities and which leads to an SCCmecC-orfX PCR product of 224 bp. As summarized in Table 2, supplementation of the MRSA-screening PCR (3) with this primer enabled detection of the mecC-positive MRSA strains Sc26644 and Sc16837, of a mecC-carrying MRSA strain analyzed for external quality control, and of seven animal S. aureus strains harboring mecC (5) without affecting the detection of MRSA strains of SCCmec types I to VI. Fifty MRSA isolates from our strain collection (including 12 different spa types) and 50 methicillin-susceptible S. aureus isolates (consecutively collected from routine specimens) were tested in the SCCmec XI-supplemented MRSA-screening PCR, revealing 100% sensitivity and 100% specificity. Fifty consecutive nasopharyngeal swabs tested MRSA positive in routine screening diagnostics, and 50 consecutive nasopharyngeal swabs with MRSA-negative results in routine testing were additionally analyzed. No difference in mecA-carrying MRSA detection compared to the original PCR (3) was observed, indicating that the robustness of the MRSA-screening PCR is not affected by addition of the SCCmec XI primer. It should be noted that the test may perform differently in different clinical settings due to different population characteristics.

This is, to our knowledge, the first description of an SCCmec XI primer which now allows the rapid detection of mecC-positive MRSA by MRSA-screening real-time PCR without former cultivation of MRSA directly from nasopharyngeal swabs.