Infect Chemother.  2011 Oct;43(5):396-405. 10.3947/ic.2011.43.5.396.

Application of Infrequent-Restriction-Site Polymerase Reaction (IRS-PCR) to the Molecular Epidemiologic Analysis of Methicillin Resistant Staphylococcus aureus (MRSA)

• Shin NY
• Yoo JH
• Park C
• Lee DG
• Choi SM
• Kwon JC
• Kim SH
• Park SH
• Choi JH

• KMID: 2285012
• DOI: http://doi.org/10.3947/ic.2011.43.5.396

Abstract

BACKGROUND
We investigated the usefulness of infrequent-restriction-site polymerase chain reaction (IRS-PCR) compared with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) on the molecular epidemiologic analysis of methicillin-resistant Staphylococcus aureus (MRSA).
MATERIALS AND METHODS
We used fifty clinical isolates of MRSA collected from 10 university hospitals located in Seoul. We performed three procedures on these isolates: PFGE using SmaI, IRS-PCR using XbaI-Hha I or EagI-Hha I, and MLST using seven house-keeping genes. We determined the clusters of molecular types by dendrogram using the unweighted pair group method with arithmetic mean (UPGMA) and Dice coefficients.
RESULTS
MLST analysis showed that isolates exhibited ST1, ST5, ST72, ST89, and ST239. In PFGE, the isolates clustered into 5 major groups with 80% similarity, which subsequently became classified into 18 subgroups with 95% similarity. In IRS-PCR using EagI-HhaI restriction enzymes, there was little resolution among the patterns of isolates. However, XbaI-HhaI IRS-PCR showed 5 groups with a 90% similarity. These groups were then classified into 9 subgroups with a 95% similarity. There were no significant differences among the isolates from different hospitals.
CONCLUSIONS
The XbaI-HhaI IRS-PCR method could be a useful tool in the molecular epidemiology of MRSA. Its resolution power was good enough to analyze isolates, because the patterns of IRS-PCR were closely correlated with those of MLST and showed diverse groups.

Keyword

Methicillin resistant Staphylococcus aureus; Pulsed Field Gel Electrophoresis; Infrequent restriction site PCR; Multilocus sequence typing

MeSH Terms

Electrophoresis, Gel, Pulsed-Field
Genes, Essential
Hospitals, University
Methicillin
Methicillin Resistance
Methicillin-Resistant Staphylococcus aureus
Molecular Epidemiology
Multilocus Sequence Typing
Polymerase Chain Reaction
Staphylococcus
Staphylococcus aureus
Methicillin

Figure

• 

  Figure 1 Schematic presentation of IRS-PCR.

• 

  Figure 2 IRS-PCR electrophoretic patterns of MRSA produced by amplification of restricted-ligated XbaI-HhaI fragments. (A) Lane 1 and 10 contained 25-bp ladders; lane 2 through 9 contained clinically isolated strains using adaptor (AH1&AH2, AX1 & AX2, AE1 & AE2) and primer PX-C. (B) Lane 1, 8, and 15 contained 25-bp ladders; lane 2 through 7 and 9 through 14 contained clinically-isolated strains using new adaptor (AH1 & AH2-new, AX1 & AX2-new, AE1 & AE2-new) and primer PX-C. (C) Lane 1 and 10 contained 25-bp ladders; lane 2 through 9 contained clinically-isolated strains using new adaptor (AH1 & AH2-new, AX1 & AX2-new, AE1 & AE2-new) and primer PX-CG.

• 

  Figure 3 Representative products of PFGE. Lane 1, 7, 13, and 20 contained DNA marker (NCTC8325 S. aureus); lane 2 through 6, 8 through 12, and 14 through 19 contained clinically-isolated strains.

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  Figure 4 IRS-PCR electrophoretic patterns of MRSA produced by amplification of restricted-ligated XbaI-HhaI DNA using primer PX-CG in MRSA. The lane 1 in each gel contained 25-bp ladders; (A) ST239 isolates. (B) lane 2 to 5 contained ST89 isolates; lanes 6 to 9 contained ST72 isolates. (C) ST1 isolates. (D) ST5 isolates.

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  Figure 5 Dendrogram of MRSA in PFGE.

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  Figure 6 Dendrogram of MRSA in IRS-PCR.

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