Power Of Pulsed-field Gel Electrophoresis Research Articles

Comparison of Conventional Molecular and Whole-Genome Sequencing Methods for Differentiating Salmonella enterica Serovar Schwarzengrund Isolates Obtained from Food and Animal Sources.

Over the last decade, Salmonella enterica serovar Schwarzengrund has become more prevalent in Asia, Europe, and the US with the simultaneous emergence of multidrug-resistant isolates. As these pathogens are responsible for many sporadic illnesses and chronic complications, as well as outbreaks over many countries, improved surveillance is urgently needed. For 20 years, pulsed-field gel electrophoresis (PFGE) has been the gold standard for determining bacterial relatedness by targeting genome-wide restriction enzyme polymorphisms. Despite its utility, recent studies have reported that PFGE results correlate poorly with that of closely related outbreak strains and clonally dominant endemic strains. Due to these concerns, alternative amplification-based molecular methods for bacterial strain typing have been developed, including clustered regular interspaced short palindromic repeats (CRISPR) and multilocus sequence typing (MLST). Furthermore, as the cost of sequencing continues to decrease, whole genome sequencing (WGS) is poised to replace other molecular strain typing methods. In this study, we assessed the discriminatory power of PFGE, CRISPR, MLST, and WGS methods to differentiate between 23 epidemiologically unrelated S. enterica serovar Schwarzengrund isolates collected over an 18-year period from distinct locations in Taiwan. The discriminatory index (DI) of each method for different isolates was calculated, resulting in values between 0 (not discriminatory) and 1 (highly discriminatory). Our results showed that WGS has the greatest resolution (DI = 0.982) compared to PFGE (DI = 0.938), CRISPR (DI = 0.906), and MLST (DI = 0.463) methods. In conclusion, the WGS typing approach was shown to be the most sensitive for S. enterica serovar Schwarzengrund fingerprinting.

Feasibility of Using Multiple-Locus Variable-Number Tandem-Repeat Analysis for Epidemiology Study of Vibrio parahaemolyticus Infections

Vibrio parahaemolyticus causes foodborne gastroenteritis, which is often associated with the consumption of raw or undercooked shellfish. Molecular typing can provide critical information for detecting outbreaks and for source attribution. In this study, we describe the development and evaluation of an optimized multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) for the characterization of V. parahaemolyticus isolates. The discriminatory power of MLVA was compared to that of pulsed-field gel electrophoresis (PFGE) by typing 73 sporadic isolates. Epidemiologic concordance was evaluated by typing 23 isolates from five epidemiologically well-characterized outbreaks. The optimized MLVA was applied in early warning, epidemiological surveillance, and source tracking for V. parahaemolyticus infections. There was no significant difference in the discriminatory power of PFGE and MLVA with six or eight VNTR loci for the sporadic isolates. All isolates within an outbreak were indistinguishable by MLVA with six loci, except for one outbreak. Typically, the epidemiological survey could be initiated according to PFGE clusters. We applied MLVA with six loci on 22 isolates in two PFGE clusters. Isolates in one PFGE cluster were distinguished by MLVA. Although a follow-up investigation showed that both clusters had no epidemiological concordance, MLVA decreased the frequency of initiation of epidemiological surveys, thereby reducing labor costs. The ability of MLVA to trace the source of infection was evaluated by isolates from two outbreaks and shrimp samples. The isolates from one of outbreaks and a shrimp had the same MLVA type, suggesting that an epidemiological survey was initiated. Data from the epidemiological investigation subsequently indicated that contaminated shrimp from a nearby city (Dongguan) might be the source of the outbreak. In conclusion, these results indicate that the optimized MLVA may be a promising tool for early warning and epidemiological surveillance of V. parahaemolyticus infections.

Genotyping of Yersinia enterocolitica biotype 1A strains from clinical and nonclinical origins by pulsed-field gel electrophoresis.

Yersinia enterocolitica biotype 1A (B1A) strains are considered mainly nonpathogenic. However, some studies considered strains of this biotype to be the causal agents of infections in humans and animals. In South America, there are no studies that have compared clinical and nonclinical strains of B1A typed by pulsed-field gel electrophoresis (PFGE) and none that have compared the capability of different enzymes on typing these strains. This study typed 51 Y. enterocolitica B1A strains isolated in Brazil and Chile by PFGE, testing the enzymes XbaI, NotI, and XhoI. The resulting dendrograms discriminated the strains in 47, 40, and 49 pulsotypes generated by the cleavage with the enzymes XbaI, NotI, and XhoI, respectively. The majority of the strains were grouped independently of their clinical or nonclinical origins. The high discriminatory power of PFGE confirmed the heterogeneity of B1A strains but could not divide the strains studied into clusters that differed in the frequency of some virulence genes as observed in studies using other methodologies.

Characterization ofEscherichia coliO157:H7 in New Zealand using multiple-locus variable-number tandem-repeat analysis

Recently, multiple-locus variable-number tandem-repeat analysis (MLVA) has been proposed as an alternative to pulsed-field gel electrophoresis (PFGE) for characterization of Escherichia coli O157:H7. In this study we characterized 118 E. coli O157:H7 isolates from cases of gastrointestinal disease in New Zealand using XbaI PFGE profiles and a MLVA scheme that assessed variability in eight polymorphic loci. The 118 isolates characterized included all 80 E. coli O157:H7 referred to New Zealand's Enteric Reference Laboratory in 2006 and 29 phage-type 2 isolates from 2005. When applied to these isolates the discriminatory power of PFGE and MLVA was not significantly different. However, MLVA data may be more epidemiologically relevant as isolates from family clusters of disease had identical MLVA profiles, even when the XbaI PFGE profiles differed slightly. Furthermore, most isolates with indistinguishable XbaI PFGE profiles that did not appear to be epidemiologically related had distinct MLVA profiles.

Genotyping of Campylobacter spp. from retail meats by pulsed-field gel electrophoresis and ribotyping

To determine the genetic relatedness of Campylobacter spp. from retail meat products, and compare the discriminatory power of pulsed-field gel electrophoresis (PFGE) and automatic ribotyping. A total of 378 Campylobacter isolates recovered from 159 raw meats (130 chicken, 25 turkey, three pork and one beef) sampled from 50 retail grocery stores of four supermarket chains in the Maryland suburban area from August 1999 to July 2000 were analysed by PFGE with SmaI, 120 isolates of which were also characterized by ribotyping with PstI using RiboPrinter system. A total of 148 unique PFGE patterns were identified, 91 of which were present in multiple Campylobacter isolates and 24 in multiple meat samples. Nineteen Campylobacter clones with identical PFGE patterns recurred frequently (up to nine times) throughout the sampling period. Comparing ribotyping with PFGE, we identified 44 PFGE patterns and 22 RiboGroups among the 120 isolates tested. Multiple PFGE patterns within one RiboGroup were commonly observed, as well as multiple RiboGroups within one PFGE pattern. Although Campylobacter present in retail meats were genetically diverse, certain clones persisted in poultry meats. PFGE had a greater discriminatory power than ribotyping, and the two methods were complementary in genotyping Campylobacter. Genomic DNA fingerprinting of Campylobacter confirmed diverse and recurrent Campylobacter clones in the retail meats, which provides additional data for a better understanding of the epidemiological aspect of Campylobacter infection.

Evaluation of the discriminatory power of pulsed-field gel electrophoresis and PCR fingerprinting for epidemiologic typing of Candida species.

OBJECTIVE: To evaluate the discriminatory power of genotyping methods (PCR fingerprinting and pulsed-field gel electrophoresis) validated for Candida albicans in other Candida species. METHODS: Molecular typing methods are increasingly being applied for studies where the interpretation of data essentially relies on the typing results rather than epidemiologic data. In this situation, the discriminatory power (ability to identify differences among epidemiologically unrelated strains) of the typing method is important in allowing one to draw valid conclusions. By applying PCR fingerprinting, electrophoretic karyotyping, and restriction fragment endonuclease analysis using standard restriction enzymes and primers proven to be useful in previous studies, we evaluated whether the use of multiple genotyping methods is sufficient to delineate known unrelated strains among seven Candida species. RESULTS: All three methods identified individual genotypes for each of the seven Candida species studied. However, optimal strain delineation required the combined use of all three typing methods and was observed only within the small number of C. albicans and C. tropicalis isolates tested in this study. CONCLUSION: Typing assays that are able to delineate a certain Candida species may not be used blindly for other species of that genus. Regarding the limited number of strains tested, further validation of the discriminative power of genotyping methods (including in C. tropicalis) should be done.

Discriminatory power and usefulness of pulsed-field gel electrophoresis in epidemiological studies of Pseudomonas aeruginosa

We assessed the discriminatory power of pulsed-field gel electrophoresis (PFGE) for the analysis of DNA restriction fragment length polymorphism (RFLP) in Pseudomonas aeruginosa. We determined DraI PFGE-RFLP of DNA of unrelated clinical and environmental strains, and clinical strains isolated from two intensive care units of the Besançon University Hospital. The typeability and reproducibility was 100%. The discriminatory index was 0·998, and the DNA patterns were stable in vitro and in vivo. There was a very low correlation between PFGE-RFLP and traditional typing methods. The typeability, reproducibility, the high discriminatory power and the stability of PFGE-RFLP make this a valuable method to be used in conjunction with serotyping. Further standardization and quantitative interpretation are possible and should lead to this technique becoming a library typing system.

Cytologically based physical maps of the group-2 chromosomes of wheat

We have constructed cytologically based physical maps (CBPMs), depicting the chromosomal distribution of RFLP markers, of the group-2 chromosomes of common wheat (Triticum aestivum L. em Thell). Twenty-one homozygous deletion lines for 2A, 2B, and 2D were used to allocate RFLP loci to 19 deletion-interval regions. A consensus CBPM was colinearily aligned with a consensus genetic map of group-2 chromosomes. The comparison revealed greater frequency of recombination in the distal regions. Several molecularly tagged chromosome regions were identified which may be within the resolving power of pulsed-field gel electrophoresis. The CBPMs show that the available probes completely mark the group-2 chromosomes, and landmark loci for sub-arm regions were identified for targeted-mapping.