Infect Chemother.  2012 Feb;44(1):5-10. 10.3947/ic.2012.44.1.5.

Molecular Epidemiologic Analysis of Community-Onset Extended Spectrum Beta-Lactamase (ESBL) Producing Escherichia coli Using Infrequent-Restriction-Site Polymerase Chain Reaction (IRS-PCR) with Comparison by Pulsed-Field Gel Electrophoresis (PFGE)

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

• KMID: 1782391
• DOI: http://doi.org/10.3947/ic.2012.44.1.5

Abstract

BACKGROUND
We evaluated the ability of infrequent restriction site-polymerase chain reaction (IRS-PCR) to perform molecular epidemiologic analysis of Community-Onset Extended Spectrum Beta-Lactamase (ESBL) producing Escherichia coli, and also assessed the use of PFGE as an alternative method.
MATERIALS AND METHODS
IRS-PCR assay was performed using combinations of adaptors for XbaI and HhaI restriction sites on clinical isolates of E. coli (n=51). We compared the discriminatory power, quality and efficiency of IRS-PCR to PFGE.
RESULTS
In E. coli, PFGE discriminated 39 (76.4%) and IRS-PCR discerned 41 (80.3%) of the total 51 strains. It took much less time to complete IRS-PCR (one day) than PFGE (at least 4 days).
CONCLUSIONS
IRS-PCR is a more sensitive and rapid alternative to PFGE for molecular epidemiologic analysis of E. coli.

Keyword

ESBL-producing E. coli; Pulsed-field gel electrophoresis (PFGE); Infrequent restriction site-polymerase chain reaction (IRS-PCR); Genotyping

MeSH Terms

beta-Lactamases
Electrophoresis, Gel, Pulsed-Field
Escherichia
Escherichia coli
Polymerase Chain Reaction
beta-Lactamases

Figure

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  Figure 1 Representative Pulsed-Field Gel Electrophoresis (PFGE) images of ESBL Escherichia coli isolates. Lanes 3 to 6 and 8 to 11 were BL430, BL435, BL440, BL441, BL442, BL443, BL446, BL447, BL448, BL450, and BL451, respectively. Markers were in lanes 1, 7 and 14 (50-1,000kb ladders).

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  Figure 2 Electrophoretic results of IRS-PCR amplification of restricted-ligated XbaI-HhaI fragments from ESBL strains. (A) Lane 1 was 100-bp ladder. Lanes 2 through 5 were isolates of BL99, BL101, BL102 and BL105, respectively. Clinically isolated strains used adaptors (AH1 and AX2) and primer PX-G. Tm; 20.6℃, 14.3℃. (B) Lane 1 was 100-bp ladder. Lanes 2 through 5 were isolates of BL99, BL101, BL102 and BL105, respectively. Clinically isolated strains used adaptors (AH1 and AH2) and primer PX-G. Tm; 32.6℃, 27.3℃. (C) Lane 1 was 100-bp ladder. Lanes 2 through 5 were isolates of BL99, BL101, BL102, and BL105, respectively. Clinically isolated strains used adaptors (AH1_new and AH2_new) and primer PX-G. Tm; 51.4℃, 37.4℃. (D) Representative IRS-PCR electrophoresis patterns of eight ESBL E. coli isolates. Lanes 2 through 9 were BL99, BL101, BL102, BL105, BL107, BL114, BL116 and BL117, respectively. Lanes 1 and 10 were 100bp ladders. The IRS-PCR annealing temperature was 58℃, and the primers were PX-G, AH2_new and AX2_new.

• 

  Figure 3 IRS-PCR electrophoretic patterns of ESBL E.coli produced by amplification of restricted-ligated XbaI-HhaI fragments. (A) Lane 1 and 10 were 100bp ladders. Lanes 2 through 9 were isolates BL99, BL101, BL102, BL105, BL107, BL114, BL116 and BL117, respectively. Clinically isolated strains used adaptors (AH2_new and AX2_new) and primer PX-G. Room temperature ligation performed at 20-25℃. (B) Lane 1 and 10 were 100bp ladders. Lanes 2 through 9 were isolates BL99, BL101, BL102, BL105, BL107, BL114, BL116 and BL117, respectively. Clinically isolated strains used adaptors (AH2_new and AX2_new) and primer PX-G. Room temperature ligation performed at -16℃.

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  Figure 4 Dendrogram resulting from cluster analysis of E. coli using IRS-PCR and PFGE.

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