Rapid Detection of the Pandemic Methicillin-Resistant Staphylococcus aureus Clone ST 239, a Dominant Strain in Asian Hospitals

Abstract

We describe and validate a novel PCR assay to detect the pandemic hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) lineage ST 239. Results based on previously uncharacterized isolates from a hospital in northeast Thailand support the view that at least 90% of HA-MRSA isolates in mainland Asia correspond to ST 239 or close relatives.Methicillin-resistant Staphylococcus aureus (MRSA) strains constitute a considerable health and resource burden in the hospital environment. Although there is currently a paucity of data (17), the evidence available suggests a high frequency of methicillin resistance throughout mainland Asia (9,14). Two recent papers have used multilocus sequence typing (MLST) to characterize hospital-acquired MRSA (HA-MRSA) isolates from nine Asian countries from Saudi Arabia to the Philippines (4, 15). These data suggest that at least 90% of the cases of HA-MRSA within a region accounting for Ͼ60% of the world's population can be accounted for by a single clonal subgroup, ST 239. This genotype has also been detected in 26 countries outside of Asia, corresponding to the EMRSA-1, -4, -7, -9, and -11, Brazilian (1), Portuguese, Hungarian (8), and Viennese clones, although in many countries, it has recently been replaced by other clones (1,5,7,8,13,16,(18)(19)(20).The global dissemination of this clone is consistent with heightened transmissibility, and two recent reports noted an association between ST 239 and increased virulence (2, 10). ST 239 has evolved through a large-scale recombination event involving the import of ϳ20% of the genome from CC 30 (ST 30-like), while the remaining 80% (genomic backbone) corresponds to CC 8 (ST 8-like) (21). As these two lineages are unrelated (6), this mosaic structure provides the means to develop a rapid, accurate, and sensitive PCR-based assay. We designed two pairs of discriminatory nonredundant primers based on two variable genes, one of which (SA2003) lies within the ST 8-like backbone, while the other (SA0317) lies within the ST 30-like imported region (21) (Fig. 1). As the primers were designed to amplify products of different sizes (220 bp for SA2003 and 484 bp for SA0317), it is possible to identify cases where both products are amplified from a single PCR. The presence of both amplicons of the predicted sizes indicates the presence of both the ST 30-and ST 8-like sequences in the hybrid genome of ST 239.The primer sequences used were ST 239/ST 30-like specific primers 5Ј-TCGCACTCTCGTTGAACA-3Ј (SA0317Forward) and 5Ј-AAATCCGCTTCGACAAACATT (SA0317Reverse) (Fig. 1) and ST 239/ST 8-like specific primers 5Ј-CACTTTAAATA CTGACGAAAAT-3Ј (SA2003Forward) and 5Ј-TTGAAAAT TGATCATTCAGCAA-3Ј (SA2003Reverse).Heteroduplex PCR was carried in a 25-l mixture consisting of 1ϫ PCR buffer, 1.5 mM MgCl 2 , 0.2 mM deoxynucleoside triphosphate, 0.7 M of each of the four primers, 1.25 U of HotStart Taq DNA polymerase, and 2.5 l of DNA. PCR conditions were 95°C for 15 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and 72°C for 7 min. Figu...

Comparison of Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Strains Isolated from a Tertiary Hospital in Terengganu, Malaysia

“…We performed spa and heteroduplex PCR for identification of MLST ST239 as described by Harmsen et al (12) and Feil et al (27). The spa amplicons were purified using a Qiagen DNA purification kit (Qiagen GmbH, Hilden, Germany) and sequenced to validate their identities.…”

Emergence of Methicillin-Resistant Staphylococcus aureus ST239 with High-Level Mupirocin and Inducible Clindamycin Resistance in a Tertiary Care Center in Chennai, South India

“…ethicillin-resistant Staphylococcus aureus (MRSA) ST239 is probably the most predominant clone of MRSA causing hospital-acquired infections (4). Different clones of MRSA ST239 have been reported: Hungarian, Brazilian, Portuguese, and Viennese clones (4).…”

“…Different clones of MRSA ST239 have been reported: Hungarian, Brazilian, Portuguese, and Viennese clones (4). MRSA ST239 is an invasive clone of multidrugresistant (MDR) MRSA producing potential exotoxins, causing a wide range of life-threatening infections.…”

Development of a multiplex PCR assay to detect the major clonal complexes of Streptococcus suis relevant to human infection

“…To develop a multiplex PCR assay covering the major CCs of Streptococcus suis relevant to human infections, including CC1, CC25, CC28, CC104, CC221/234 and CC233/ 379 (Feil et al, 2008, Kerdsin et al, 2011a; http://ssuis. mlst.net/), we selected hp1, mp, pep and col of the CC104-specific sequences as targets for primer design, including the srtBCD primers described by Takamatsu et al (2009).…”

“…Therefore, a PCR-based method would be an attractive alternative for determining Streptococcus suis CCs, since it can be used to screen large numbers of isolates, has rapid analytical capacity, is low cost and can be applied in most laboratories. PCR assays have been successfully developed to identify CCs in Staphylococcus aureus and Acinetobacter baumannii (Feil et al, 2008;Turton et al, 2007).…”