Rapid Pulsed-field Gel Electrophoresis Research Articles

Development of a Rapid Method for the Screening of Conjugated Linoleic Acid (CLA)-Producing Strains of Bifidobacterium breve.

This study was performed to isolate some strains of Bifidobacterium breve from fecal materials of neonates and to screen them for the biotransformation activity of converting linoleic acid into conjugated linoleic acid (CLA). Fecal samples were collected from twenty healthy neonates between 14 and 100 days old, and four hundred colonies were randomly selected from a Bifidobacterium selective transoligosaccharide medium. A duplex polymerase chain reaction technique was developed for the rapid and accurate molecular characterization of the B. breve strains that have been reported to show the species-specific characteristic of CLA production. They are identified by 16S ribosomal DNA, fructose-6-phosphate phosphoketolase encoding genes (xfp), and rapid pulsed field gel electrophoresis. Thirty-six isolates were identified as B. breve, and just two of the 12 neonates were harboring B. breve strains. Each isolate showed different CLA-producing ability in the spectrophotometric assay. All of the positive strains from the primary spectrophotometric assay were confirmed for their CLA-producing activities using gas-chromatographic analysis, and their conversion rates were different, depending on the strain isolated in this study. Some strains of B. breve were successfully isolated and characterized based on the CLA-producing activity, and further studies are necessary to characterize the enzyme and the gene responsible for the enzyme activity.

Optimization of Pulse-Field Gel Electrophoresis for Subtyping of Klebsiella pneumoniae

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.

Simple, time saving pulsed-field gel electrophoresis protocol for the typing of Stenotrophomonas maltophilia

We developed a time-saving and cost-efficient Pulsed Field Gel Electrophoresis (PFGE) method for the typing of Stenotrophomonas maltophilia by modifying the conventional procedures. Our modifications related to the cell suspension preparation, lysis of bacterial cells in plugs, washing steps, and consumption of restriction enzyme. Although few rapid PFGE protocols on Gram-negative bacteria are available, the use of comparatively large amounts of costly reagents prompted us to look for other alternative. Hence, by considering the speed, simplicity, and relatively low cost, the modified protocol may be of more practical value than other established protocols in investigating S. maltophilia nosocomial outbreaks.

A rapid PFGE protocol for typing Legionella isolates from fresh or frozen samples

Within the realm of studies on nosocomial infections and epidemiology, pulsed field gel electrophoresis (PFGE) is often considered as the "gold standard" for typing of Legionella or other bacteria. Performing this protocol usually requires 2-5 days; this excessive time requirement, lack of a standardized procedure, and high cost have limited its use. However, recently the typing of Legionella with PFGE has been reduced to about 2 days, and we further shortened the procedure by reducing the time for the plug preparation and electrophoresis steps. To shorten plug preparation, we used a strong thermal shock and high-temperature washes to reduce cell lysis and DNA isolation time, and we stressed the electrophoresis to obtain comparable macrorestriction patterns among strains in 16 h. The DNA digestion phase was not altered. We also applied this protocol directly to frozen bacteria from strain collections with the aim of shortening the entire procedure. We developed a protocol that can be completed in 24 h, or less if necessary, while avoiding some typical artifacts of traditional procedures. This new protocol also provides good results when directly applied to frozen material from strain collections, thus saving bacteria growth time. Our observations indicate that PFGE tolerates a wide range of adjustments, allowing its application to fresh or frozen samples in a short amount of time.

Evolution of tigecycline resistance inKlebsiella pneumoniaein a single patient

Carbapenemase-producing Klebsiella pneumoniae infections carry serious clinical and infection-control implications. Isolates possessing such hydrolyzing enzymes have been described in the United States and around the world. Besides being resistant to carbapenems, they usually confer resistance to fluoroquinolones, piperacillin-tazobactam, and extended-spectrum cephalosporins. Tigecycline demonstrates in vitro activity against these organisms, but reported resistance raises concern about tigecycline use for these infections. We describe a carbapenemase-producing K pneumoniae evolving resistance to tigecycline in a 75-year-old male after a prolonged stay in a critical care unit.

PulseNet USA Standardized Pulsed-Field Gel Electrophoresis Protocol for Subtyping ofVibrio parahaemolyticus

PulseNet is a national molecular subtyping network for foodborne disease surveillance composed of public health and food regulatory agencies. Participants employ molecular subtyping of foodborne pathogens using a standardized method of pulsed-field gel electrophoresis (PFGE) for conducting laboratory-based surveillance of foodborne pathogens. The PulseNet standardized PFGE protocols are developed through a comprehensive testing process. The reproducibility of the protocol undergoes an internal evaluation at the Centers for Disease Control and Prevention and an external evaluation in multiple PulseNet laboratories. Here we describe the development and evaluation of a rapid PFGE protocol for subtyping Vibrio parahaemolyticus for use in PulseNet activities. The protocol was derived from the existing standardized PulseNet protocols for Escherichia coli O157:H7 and Vibrio cholerae. An external evaluation of this protocol was undertaken in collaboration with three PulseNet USA participating public health laboratories. Comparative analysis of the PFGE fingerprints generated by each of these laboratories demonstrated that the protocol is both reliable and reproducible in the hands of multiple users.

Rapid pulsed-field gel electrophoresis protocol for subtyping of Streptococcus suis serotype 2

A rapid pulsed-field gel electrophoresis (PFGE) protocol for subtyping of Streptococcus suis serotype 2 was developed and evaluated using 27 clinical isolates from 22 epidemiologically unrelated patients. Results were matched against antibiogram, virulence genotyping and multi locus sequence typing (MLST). PFGE appeared to be the most discriminatory with numerical index of discrimination (D) equal to 0.87.

Technical Note: A Rapid Pulsed-Field Gel Electrophoresis Method for Analysis of Bifidobacteria

Pulsed-field gel electrophoresis (PFGE) is a widely used and highly discriminatory molecular typing method that has been applied to bifidobacteria. However, published PFGE protocols used with bifidobacteria require between 5 and 7 d to complete. A rapid PFGE method was developed that can be completed within 24h.

Standardization of Pulsed-Field Gel Electrophoresis Protocols for the Subtyping ofEscherichia coliO157:H7,Salmonella, andShigellafor PulseNet

Standardized rapid pulsed-field gel electrophoresis (PFGE) protocols for the subtyping of Escherichia coli O157:H7, Salmonella serotypes, and Shigella species are described. These protocols are used by laboratories in PulseNet, a network of state and local health departments, and other public health laboratories that perform real-time PFGE subtyping of these bacterial foodborne pathogens for surveillance and outbreak investigations. Development and standardization of these protocols consisted of a thorough optimization of reagents and reaction conditions to ensure that the protocols yielded consistent results and high-quality PFGE pattern data in all the PulseNet participating laboratories. These rapid PFGE protocols are based on the original 3-4-day standardized procedure developed at Centers for Disease Control and Prevention that was validated in 1996 and 1997 by eight independent laboratories. By using these rapid standardized PFGE protocols, PulseNet laboratories are able to subtype foodborne pathogens in approximately 24 h, allowing for the early detection of foodborne disease case clusters and often aiding in the identification of the source responsible for the infections.

Development and Validation of a PulseNet Standardized Pulsed-Field Gel Electrophoresis Protocol for Subtyping ofVibrio cholerae

PulseNet is a network that utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols with the purpose of conducting laboratory-based surveillance of foodborne pathogens. PulseNet standardized PFGE protocols are subject to rigorous testing during the developmental phase and careful evaluation during a validation process assessing its robustness and reproducibility in different laboratories. Here we describe the development and validation of a rapid PFGE protocol for subtyping Vibrio cholerae for use in PulseNet International activities. While the protocol was derived from the existing PulseNet protocol for Escherichia coli O157, various aspects of this protocol were optimized for use with V. cholerae, most notably a change of the primary and secondary restriction enzyme to SfiI and NotI, respectively, and the use of a two-block electrophoresis program. External validation of this protocol was undertaken through a collaboration between three PulseNet Asia Pacific laboratories (Public Health Laboratory Centre, Hong Kong, National Institute of Infectious Diseases, Japan, and International Center for Diarrhoeal Diseases Research-Bangladesh) and PulseNet USA. Comparison of PFGE patterns generated by each of the participating laboratories demonstrated that the protocol is robust and reproducible.

An Outbreak Due to Serratia marcescens in a Neonatal Intensive Care Unit Typed by 2-Day Pulsed Field Gel Electrophoresis Protocol

Background. Serratia marcescens is a well-recognized nosocomial pathogen. The objective of the study was to describe typing results using a rapid pulsed field gel electrophoresis (PFGE) protocol and infection control measures during an outbreak of Serratia marcescens in a 24-bed, referral, neonatal intensive care unit (NICU) of a tertiary-care pediatric hospital. Methods. Two patients with S. marcescens sepsis were identified in the NICU. Health care personnel of the unit were requested to reinforce infection control measures. Active surveillance was established to detect infected and/or colonized patients and environmental and staff reservoirs. Infected and colonized patients were cohorted on one side of the unit; admissions to NICU were limited. Isolates were typed with a short 2-day pulsed-field gel electrophoresis (PFGE) protocol. Results. Thirty three patients were exposed during a period of 20 days. Ten S. marcescens isolates were obtained from six patients, in two from blood culture and in three from stool culture; a single clone was identified in four. S. marcescens was not isolated from environmental or staff cultures. Conclusions. PFGE results were obtained in 2 days, infection control measures were reinforced, outbreak was promptly interrupted, and the NICU remained opened.

Rapid pulsed-field gel electrophoresis technique for determination of genetic diversity of Serratia marcescens.

Seventeen Serratia marcescens strains were isolated from patients admitted to a hospital in the Tokyo metropolitan area. In order to study their genetic diversity, pulsed-field gel electrophoresis (PFGE) was performed. Clear bands were not observed by the standard PFGE technique following the instruction manual from the company. To obtain clear results, it was necessary to include some additional steps to the preparation of PFGE samples. We added an inactivation step for DNase using formaldehyde, and the bacterial strains were embedded in thinner plug molds than usual. These modifications were effective with all strains, and the complete PFGE pattern was obtained in the samples treated with formaldehyde and using thin plug molds. We established a rapid method to obtain a high-quality PFGE result for S. marcescens.

Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram-negative organisms in 1 day.

Genomic DNA patterns generated by pulsed-field gel electrophoresis are highly specific for different strains of an organism and have significant value in epidemiologic investigations of infectious-disease outbreaks. Unfortunately, time-consuming and tedious specimen processing is an inherent problem which limits the use of this powerful technology as a real-time epidemic investigational tool. Here, I describe a rapid method to improve the response time and provide specific bacterial strain identification for the typing of Escherichia coli O157:H7 and other gram-negative organisms in a single day.