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)

Ji-Hyun Byun; Jin-Hong Yoo; Chulmin Park; Dong-Gun Lee; Sun-Hee Park; Su-Mi Choi; Jung-Hyun Choi; Si-Hyun Kim; Jae-Cheol Kwon

doi:10.3947/ic.2012.44.1.5

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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)

Original Article

Infection & Chemotherapy

Infection & Chemotherapy 2012; 44(1): 5-10.

Published online: 29 February 2012

DOI: https://doi.org/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)

Ji-Hyun Byun¹, Jin-Hong Yoo², Chulmin Park³, Dong-Gun Lee²

, Sun-Hee Park², Su-Mi Choi², Jung-Hyun Choi², Si-Hyun Kim², Jae-Cheol Kwon²

¹Department of Biomedical Science, The Catholic University of Korea, Graduate School, Seoul, Korea.

²Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea.

³Catholic Research Institutes of Medical Science, The Catholic University of Korea, Seoul, Korea.

Correspondence to Jin-Hong Yoo. Division of Infectious Disease, Department of Internal Medicine, Bucheon St. Mary's Hospital, 2 Sosa-dong, Wonmi-gu, Bucheon 420-717, Korea. Tel: +82-1577-0675, Fax: +82-32-340-2669, jhyoo@catholic.ac.kr

Received 7 July 2011 Revised 14 December 2011 Accepted 11 January 2012

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

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

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).

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℃.

Figure 4

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

Table 1

Adaptors and Primers used in the Infrequent-Restriction-Site Polymerase Chain Reaction

^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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ORCID iDs: Dong-Gun Lee
https://orcid.org/http://orcid.org/0000-0003-4655-0641
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