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Volume 46, Number 3, June 2013

Suitable restriction enzyme for standardization of pulsed-field gel electrophoresis protocol and interlaboratory comparison of Acinetobacter baumannii 


Kai-Ming Chang, Wei-Chang Huang, Chien-Shun Chiou, Gwan-Han Shen, Chen-Cheng Huang, Fu-Shyan Wen


Received: March 14, 2012    Revised: April 13, 2012    Accepted: July 23, 2012   

 

Corresponding author:

Department of Life Science, National Chung-Hsing University, Taichung, Taiwan, ROC
Corresponding author. Department of Life Sciences, National Chung-Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan, ROC 



 

Background and purpose: 

Interlaboratory comparison of pulsed-field gel electrophoresis (PFGE) patterns is difficult. A key reference of standardized PFGE protocol for Acinetobacter baumannii may address this issue. This study aimed to determine restriction enzymes with rare cutting sites on A baumannii genomes and evaluate their cost-effectiveness, discriminatory power, and interlaboratory consistence of band assignments. 



 

Methods:

There were 42 A baumannii isolates collected, including nine from three hospital outbreaks and 33 sporadic isolates. The numbers of cutting sites for the restriction enzymes were explored using the “Restriction Digest and PFGE” program. The cost-effectiveness for PFGE analysis was evaluated for the tested restriction enzymes, while its discriminatory ability was expressed through a discriminatory index and 95% confidence interval. The interlaboratory consistence of band assignments was evaluated for the 42 A baumannii isolates. 



 

Results:

ApaI was the most cost-effective restriction enzyme for a PFGE protocol for A baumannii. Both AscI and AsiSI were reasonable in terms of costs. ApaI, AscI, and AsiSI exhibited similar discriminatory indices. ApaI generated more than 40 fragments that were close and not easy to resolve, resulting in less consistence of band assignments. AscI and AsiSI generated 10–20 fragments that were clearly resolved, resulting in higher consistence of band assignments. AscI exhibited a close discriminatory power to that of AsiSI and at half of the cost of AsiSI for PFGE analysis. 



 

Conclusion:

We recommend AscI as the primary enzyme and AsiSI as the secondary enzyme for standardizing the PFGE protocol and interlaboratory comparisons of A baumannii. 



 

Key words:

Acinetobacter baumannii, AscI, AsiSI, Pulsed-field gel electrophoresis, Restriction enzyme