Journal List > Infect Chemother > v.44(1) > 1035210

Byun, Yoo, Park, Lee, Park, Choi, Choi, Kim, and Kwon: 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)

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.

Figures and Tables

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.
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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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Table 1
Adaptors and Primers used in the Infrequent-Restriction-Site Polymerase Chain Reaction
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aNewly designed for this study.

Table 2
Results of PFGE and IRS-PCR Performed on 51 Clinical Isolates of E. coli
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Dong-Gun Lee
https://orcid.org/http://orcid.org/0000-0003-4655-0641

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