The PulseNet standardized PFGE protocols were adopted by the laboratories of Taiwan Centers for Disease Control (Taiwan CDC (external link) ) in 2002 for routine subtyping and building a DNA fingerprint database of bacterial pathogens. After 4-year practice, PulseNet Taiwan, the National Molecular Subtyping Network for Infectious Disease Surveillance, was formally inaugurated by the Department of Health, Taiwan, on 3 October 2006.
Foodborne pathogens included in PulseNet Taiwan (up to 2007)
|Bacterial species||Number of isolates||Number of PFGE patterns|
|Vibro cholerae (most are reference strains)||26||19|
|E. coli O157||12||11|
Salmonellosis is estimated to be the most prevalent foodborne disease. To monitor this disease, a National Salmonella reference laboratory was established by Taiwan CDC in 2004. More than 2,000 Salmonella isolates were collected each year from the collaborative hospitals for determination of serotypes, PFGE fingerprints and traits of antimicrobial susceptibility. To date, the Salmonella DNA fingerprint database has contained more than 10,000 PFGE fingerprint entries representing for over 70 serotypes. For some serotypes with abundant PFGE genotypes, the serotype of a new isolate can be predicted by comparison of PFGE fingerprint with those in the fingerprint database. Since 2007, we have applied this database for serotype prediction and serotypes of 98.44% (3,034/3,082) isolates of the year were determined by PFGE fingerprint comparison. Taiwan PulseNet has been applied to confirm an international outbreak of Salmonella Agona infections linked to infant food and rule out many salmonellosis outbreaks occurred in USA.
S. flexneri is mainly circulating among aboriginal tribes in the mountainous area; while S. sonnei infections mainly occurs in the industrialized western Taiwan and most are associated with imported clones. All the Shigella isolates are sent to the laboratories of Taiwan CDC and routinely analyzed by PFGE with NotI and XbaI. S. sonnei isolates are also routinely analyzed by MLVA method. The Shigella DNA fingerprint database has been used to rule out some international shigellosis outbreaks including the recent outbreak linked to baby corn exported from Thailand.
V. parahaemolyticus is the leading causal agent for foodborne disease outbreaks in Taiwan. Since most of the outbreaks of V. parahaemolyticus infections are easily identified, routine PFGE analysis is not applied to this organism.
To date, Taiwan has no indigenous case of E. coli O157:H7 infection; only an imported case was identified in 2001. V. cholerae infection was rare for the past 40 years. Listeriosis is not in the list of notifiable diseases in Taiwan. Although the incidence rate is unknown, L. monocytogenes is infrequently identified from patients in this country.
Other bacterial pathogens included in the DNA fingerprint database
|Bacterial species||Number of Isolates||Number of PFGE patterns|
The PulseNet platform is also applied to build a DNA fingerprint database for some infrequent as foodborne or non-foodborne bacterial pathogens. Streptococcus pyogenes DNA fingerprint database contains PFGE fingerprints and emm sequences. Neisseria meningitidis database contains PFGE fingerprints, MLVA profiles, and MLST, proA and proB sequences; whereas, Bordetella pertussis database contains only PFGE fingerprints of the organism.
PFGE Technique Development
An automatic plug washer has been invented by a local company, which was an improved model of the primordial plug washer originally designed by US CDC. The quality of DNA prepared by using the new plug washer is good and stable. We also evaluated the restriction enzyme and protocol for PFGE analysis of V. parahaemolyticus. The study indicated that the PulseNet standardized PFGE protocol for enterobacteria with some modifications is applicable to PFGE analysis of V. parahemoltyicus and NotI is more cost-effective than SfiI. Our data indicated that NotI should be used as the primary enzyme and SfiI as the second enzyme for PFGE analysis of V. parahaemolyticus.
Development of next generation of molecular subtyping method
With grant support partly from the National Institute of Infectious Diseases, Japan, Taiwan CDC has successfully developed a MLVA method with 26 VNTRs for S. sonnei. The MLVA method exhibits higher level of discriminatory power than PFGE. The method is potentially a useful tool for phylogenetic analysis of S. sonnei. Development of MLVA methods for S. flexneri and Salmonella Typhimurium are one of the current focuses of Taiwan CDC.
PFGE and genotyping capacity in Taiwan CDC's laboratories
The bacterial laboratories of Taiwan CDC have been equipped with 11 PFGE instruments (Bio-Rad CHEF Mapper), 3 sequencers (ABI 3130) and more than 10 sets of BioNumerics software. PFGE analysis of some bacterial pathogens is routinely performed in the laboratories. Since 2004, more than 3,000 bacterial isolates were analyzed each year. With the aid of automatic plug washer, the PFGE analysis capacity of the laboratories can be up to 8,000 isolates per year.
PulseNet Taiwan has been adopted by DOH as an early detection tool in the national food safety surveillance system. In this regard, the PulseNet Taiwan Task Force will include partners from food and agriculture sectors. In the future, PulseNet Taiwan will focus on the surveillance of shigellosis and to be an active member in the PulseNet International community.
For further information please contact:
Dr Chien-Shun Chiou, Centers for Disease Control, Taiwan.
PulseNet Asia Pacific group photo, Thailand, December 2009