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. 2013 Jul 1;10(7):2720-31.
doi: 10.3390/ijerph10072720.

Optimization of pulse-field gel electrophoresis for subtyping of Klebsiella pneumoniae

Hui Han  1 , Haijian Zhou, Haishan Li, Yuan Gao, Zhi Lu, Kongxin Hu, Baoliang Xu
Affiliations

Optimization of pulse-field gel electrophoresis for subtyping of Klebsiella pneumoniae

Hui Han et al. Int J Environ Res Public Health. .

Abstract

A total of 110 strains of Klebsiella pneumoniae were used to optimize pulsed-field gel electrophoresis (PFGE) for subtyping of K. pneumoniae. For optimization of electrophoresis parameters (EPs) of XbaI-PFGE, 11 isolates were analyzed with XbaI digestion using three EPs. The EP of a switch time of 6 to 36 s for 18.5 h gave clearest patterns and was declared the optimal EP for XbaI PFGE of K. pneumoniae. By software analysis and pilot study, AvrII was chosen as another PFGE enzyme. Both XbaI- and AvrII-PFGE gave D-values higher than 0.99 for 69 K. pneumoniae isolated from different sources. Our results also showed good typeability, reproducibility of both XbaI- and AvrII-PFGE for K. pneumoniae subtyping. Furthermore, the established PFGE method also had good discriminatory power to distinguish outbreak K. pneumoniae strains and a high degree of consistency with multilocus sequence typing method. A rapid PFGE protocol was established here, which could be used for genotyping and other researches of K. pneumoniae.

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Figures

Figure 1
Figure 1
PFGE images obtained using three EPs with XbaI digestion. Lands 1, 5, 10, 15 were DNA size marker Salmonella serotype Braenderup H9812. Lands 2–4, 6–9, 11–14 were tested K. pneumonia.
Figure 2
Figure 2
PFGE images of four K. pneumoniae isolates restricted with AvrII (lanes 2 to 5), PmeI (lanes 7 to 10), SpeI (lanes 12 to 15), XbaI (lanes 17 to 20) and SwaI (lanes 22 to 25). The size standard (H9812) was loaded in lanes 1, 6, 11, 16, and 21.
Figure 3
Figure 3
Clustering results of patterns obtained with XbaI- and AvrII-PFGE respectively. Charts are shown for 69 K. pneumoniae strains.
Figure 4
Figure 4
Clustering results of patterns obtained with XbaI- and AvrII-PFGE of 11 K. pneumoniae strains from one outbreak.
Figure 5
Figure 5
Clustering results of patterns obtained with XbaI digestion of 32 K. pneumoniae strains tested by multilocus sequence typing.
Figure 6
Figure 6
Comparing of PFGE (XbaI digestion) and MLST to subtype K. pneumoniae strains isolated during an outbreak period.
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