Development of a rapid strain differentiation method for methicillin-resistant Staphylococcus aureus isolated in Japan by detecting phage-derived open-reading frames

Abstract

MRSA isolates can be genotyped rapidly by detecting phage-derived ORFs. As particular pandemic clones can be found in a specific region, a typing method localized to a pandemic clone may be effective for the rapid genotyping of MRSA during outbreaks.

Rapid Identification of Methicillin-Resistant Staphylococcus aureus Transmission in Hospitals by Use of Phage-Derived Open Reading Frame Typing Enhanced by Multiplex PCR and Reverse Line Blot Assay

“…Two probes (one sense and one antisense) were designed for each target. Primer modification and probe design were based on bacteriophage and gene sequences referenced in the original paper (16) and published in NCBI GenBank (Table 1). Primer and probe specificities were verified using BLASTn searches of published sequences in NCBI GenBank.…”

“…N046Fb 1746-TGATGGAGAGGAGTGGGATA-1765 X03216 N046AP 1789-AGTTACGTCTATCCCAAACGTCTT-1766 X03216 N046SP 2096-TGGAAGGACTATTTAGTACCCTTCTTA-2122 X03216 N046Rb 2143-GAACATCATCCCATTCTAGCC-2123 X03216 Mu50 SaGIm SAV0803 SAV0803Fb 881430-CAATATCCAAACCACGACCC-881411 BA000017 SAV0803AP 881376-GATGAAAAGCAAATATACATATATAACTCTACATG-881410 BA000017 SAV0803SP 881245-TCTTTTAATATTTCTTTATGATTGTATTTATTATATGTATA TG- Primer and probe design. Primers were modified from the originally published set (16) to produce a melting temperature of approximately 60°C. When necessary, additional inner primers were designed to produce amplicon lengths of Ͻ400 bp.…”

“…A range of methods for rapid identification and subtyping of methicillin-resistant Staphylococcus aureus (MRSA), which can improve the effectiveness of infection control procedures, have been reported (9,13,15,16). However, their ability to detect person-to-person transmission in settings with high-level endemicity has not been evaluated fully.…”

“…However, their ability to detect person-to-person transmission in settings with high-level endemicity has not been evaluated fully. Phage-derived open reading frame (PDORF) typing is a typing method for MRSA that was recently described from Japan and was reported to have similar discriminatory power and stability to those of pulsedfield gel electrophoresis (PFGE) (16). PDORF typing interrogates the presence or absence of 16 variable elements within the S. aureus genome: 13 from integrated prophages, 1 from a genomic island, and 2 from the SCCmec element.…”

“…PDORF typing interrogates the presence or absence of 16 variable elements within the S. aureus genome: 13 from integrated prophages, 1 from a genomic island, and 2 from the SCCmec element. In its original form, PDORF typing involves four 4-plex PCRs, each with product detection by agarose gel electrophoresis (16).…”