scholarly journals Amplified fragment length polymorphism and whole genome sequencing: a comparison of methods in the investigation of a nosocomial outbreak with vancomycin resistant enterococci

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Victoria A. Janes ◽  
Daan W. Notermans ◽  
Ingrid J.B. Spijkerman ◽  
Caroline E. Visser ◽  
Marja E. Jakobs ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Whole Genome ◽  
Nosocomial Outbreak ◽  
Outbreak Management ◽  
Sequencing Errors ◽  
Vancomycin Resistant ◽  
Random Sequencing

Abstract Background Recognition of nosocomial outbreaks with antimicrobial resistant (AMR) pathogens and appropriate infection prevention measures are essential to limit the consequences of AMR pathogens to patients in hospitals. Because unrelated, but genetically similar AMR pathogens may circulate simultaneously, rapid high-resolution molecular typing methods are needed for outbreak management. We compared amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS) during a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VRE) that spanned 5 months. Methods Hierarchical clustering of AFLP profiles was performed using unweighted pair-grouping and similarity coefficients were calculated with Pearson correlation. For WGS-analysis, core single nucleotide polymorphisms (SNPs) were used to calculate the pairwise distance between isolates, construct a maximum likelihood phylogeny and establish a cut-off for relatedness of epidemiologically linked VRE isolates. SNP-variations in the vanB gene cluster were compared to increase the comparative resolution. Technical replicates of 2 isolates were sequenced to determine the number of core-SNPs derived from random sequencing errors. Results Of the 721 patients screened for VRE carriage, AFLP assigned isolates of 22 patients to the outbreak cluster. According to WGS, all 22 isolates belonged to ST117 but only 21 grouped in a tight phylogenetic cluster and carried vanB resistance gene clusters. Sequencing of technical replicates showed that 4–5 core-SNPs were derived by random sequencing errors. The cut-off for relatedness of epidemiologically linked VRE isolates was established at ≤7 core-SNPs. The discrepant isolate was separated from the index isolate by 61 core-SNPs and the vanB gene cluster was absent. In AFLP analysis this discrepant isolate was indistinguishable from the other outbreak isolates, forming a cluster with 92% similarity (cut-off for identical isolates ≥90%). The inclusion of the discrepant isolate in the outbreak resulted in the screening of 250 patients and quarantining of an entire ward. Conclusion AFLP was a rapid and affordable screening tool for characterising hospital VRE outbreaks. For in-depth understanding of the outbreak WGS was needed. Compared to AFLP, WGS provided higher resolution typing of VRE isolates with implications for outbreak management.

scholarly journals Comparison of Whole Genome Sequencing versus Standard Molecular Diagnostics for Species Identification in the Leishmania Viannia Subgenus

2021 ◽  
Author(s):  
Rachel Lau ◽  
Avinash N. Mukkala ◽  
Ruwandi Kariyawasam ◽  
Shareese Clarke ◽  
Braulio M. Valencia ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Whole Genome Sequencing ◽  
Species Identification ◽  
Genome Sequencing ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Single Species ◽  
Whole Genome ◽  
Restriction Fragment Length

The prognosis and treatment of New World tegumentary leishmaniasis is dependent on the infecting species, yet such species identification in the Leishmania Viannia subgenus poses a diagnostic challenge. Currently, speciation relies on standard molecular techniques such as restriction fragment length polymorphism (RFLP) analysis, and Sanger sequencing (SS). Whole-genome sequencing (WGS) is a robust and increasingly cost-efficient tool that may improve Leishmania species identification. We evaluated WGS versus standard RFLP-SS for species identification in three reference and five clinical strains of Leishmania Viannia spp. Internal transcribed spacer1 (its1), cysteine proteinase b (cpb), and heat shock protein 70 (hsp70) polymerase chain reaction-restriction fragment length polymorphism (RFLP) was performed, followed by SS of the its2, cpb, hsp70, and mannose phosphate isomerase (mpi) loci. After de novo assembly, sequences were mapped, and homology compared with both reference strains and reference genomes on National Center for Biotechnology Information. All American Type Culture Collection strains were confirmed to be single-species of L. V. braziliensis, L. V. guyanensis, or L. V. panamensis by WGS. Conversely, RFLP-SS was able to definitively identify one of three isolates to the species level. Clinical samples were identified as either single-species (N = 3), mixed (N = 1), or hybrid (N = 1) infections by WGS, while standard molecular diagnosis required multi-target composite analysis for identification due to loci-dependent results by RFLP-SS. We have corroborated the utility of WGS as a diagnostic tool to speciate members of the L. Viannia subgenus and to discriminate between mixed and hybrid infections. WGS is a potentially useful complement to multistaged RFLP-SS for species identification in Leishmania infections.

scholarly journals Vancomycin-Resistant Enterococcus faecium (VREF) from Norwegian Poultry Cluster with VREF from Poultry from the United Kingdom and The Netherlands in an Amplified Fragment Length Polymorphism Genogroup

2002 ◽  
Vol 68 (6) ◽  
pp. 3133-3137 ◽  
Author(s):  
Katrine Borgen ◽  
Yngvild Wasteson ◽  
Hilde Kruse ◽  
Rob J. L. Willems
Keyword(s):  
Length Polymorphism ◽  
Genetic Relationship ◽  
Fragment Length ◽  
Enterococcus Faecium ◽  
Fragment Length Polymorphism ◽  
Amplified Fragment Length Polymorphism ◽  
Vancomycin Resistant Enterococcus ◽  
Homogeneous Population ◽  
The United Kingdom ◽  
Vancomycin Resistant

ABSTRACT The genetic relationship between 197 vancomycin-resistant Enterococcus faecium (VREF) isolates and 21 vancomycin-susceptible E. faecium isolates from Norwegian poultry was analyzed by amplified fragment length polymorphism (AFLP). The isolates were compared to 255 VREF isolates from various sources and countries. The Norwegian isolates constituted a relatively homogeneous population of E. faecium and clustered in a previously defined poultry AFLP genogroup.

scholarly journals Different Levels of Genetic Homogeneity in Vancomycin-Resistant and -Susceptible Enterococcus faecium Isolates from Different Human and Animal Sources Analyzed by Amplified-Fragment Length Polymorphism

2002 ◽  
Vol 46 (9) ◽  
pp. 2779-2783 ◽  
Author(s):  
Nienke Bruinsma ◽  
Rob J. L. Willems ◽  
Anthony E. van den Bogaard ◽  
Marga van Santen-Verheuvel ◽  
Nancy London ◽  
...  
Keyword(s):  
The Netherlands ◽  
Healthy Volunteers ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Enterococcus Faecium ◽  
Genetic Similarity ◽  
Fragment Length Polymorphism ◽  
Amplified Fragment Length Polymorphism ◽  
Hospitalized Patients ◽  
Vancomycin Resistant

ABSTRACT The genetic relationship among fecal vancomycin-resistant Enterococcus faecium (VREF) and vancomycin-susceptible E. faecium (VSEF) isolates (n = 178) from the same populations of pigs, human healthy volunteers, and hospitalized patients (from The Netherlands) and chickens (from The Netherlands and Greece) was studied by amplified-fragment length polymorphism (AFLP). The majority of VREF isolates from pigs, healthy volunteers, and hospitalized patients grouped together (genetic similarity, ≥65%). In a previous AFLP study by our group the VREF isolates from hospitalized patients grouped separately, most likely because these were clinical and not fecal isolates as in the present study. Furthermore, VSEF isolates from humans and pigs were found much more genetically diverse than VREF isolates, whereas VREF and VSEF isolates from chickens clustered together in a separate genogroup (genetic similarity, ≥65%), a pattern clearly distinct from the patterns for human and pig isolates. The present study suggests that pigs are a more important source of VREF for humans than chickens and that human- and pig-derived VSEF isolates seem much more heterogeneous than VREF isolates.

scholarly journals Whole-Genome Sequencing of Bacterial Pathogens: the Future of Nosocomial Outbreak Analysis

2017 ◽  
Vol 30 (4) ◽  
pp. 1015-1063 ◽  
Author(s):  
Scott Quainoo ◽  
Jordy P. M. Coolen ◽  
Sacha A. F. T. van Hijum ◽  
Martijn A. Huynen ◽  
Willem J. G. Melchers ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Health Care Professionals ◽  
Resistant Bacteria ◽  
Whole Genome ◽  
Nosocomial Outbreak ◽  
Icu Patients ◽  
Outbreak Management ◽  
Sequencing Technologies ◽  
Analysis Tools

SUMMARY Outbreaks of multidrug-resistant bacteria present a frequent threat to vulnerable patient populations in hospitals around the world. Intensive care unit (ICU) patients are particularly susceptible to nosocomial infections due to indwelling devices such as intravascular catheters, drains, and intratracheal tubes for mechanical ventilation. The increased vulnerability of infected ICU patients demonstrates the importance of effective outbreak management protocols to be in place. Understanding the transmission of pathogens via genotyping methods is an important tool for outbreak management. Recently, whole-genome sequencing (WGS) of pathogens has become more accessible and affordable as a tool for genotyping. Analysis of the entire pathogen genome via WGS could provide unprecedented resolution in discriminating even highly related lineages of bacteria and revolutionize outbreak analysis in hospitals. Nevertheless, clinicians have long been hesitant to implement WGS in outbreak analyses due to the expensive and cumbersome nature of early sequencing platforms. Recent improvements in sequencing technologies and analysis tools have rapidly increased the output and analysis speed as well as reduced the overall costs of WGS. In this review, we assess the feasibility of WGS technologies and bioinformatics analysis tools for nosocomial outbreak analyses and provide a comparison to conventional outbreak analysis workflows. Moreover, we review advantages and limitations of sequencing technologies and analysis tools and present a real-world example of the implementation of WGS for antimicrobial resistance analysis. We aimed to provide health care professionals with a guide to WGS outbreak analysis that highlights its benefits for hospitals and assists in the transition from conventional to WGS-based outbreak analysis.

Identification of AFLP markers on the satellite region of chromosome 1BS in wheat

Genome ◽  
2000 ◽  
Vol 43 (5) ◽  
pp. 729-735 ◽  
Author(s):  
Hangning Zhang ◽  
Shuhei Nasuda ◽  
Takashi R Endo
Keyword(s):  
Length Polymorphism ◽  
Fragment Length ◽  
Chinese Spring ◽  
Fragment Length Polymorphism ◽  
Storage Proteins ◽  
Fertility Restoration ◽  
Average Rate ◽  
Aflp Markers ◽  
Deletion Mapping ◽  
Whole Genome

The satellite region on the short arm of chromosome 1B in wheat (Triticum aestivum L., 2n = 6x = 42) carries many agronomically important genes; i.e., genes conferring fungal disease resistance, seed storage proteins, and fertility restoration. To find molecular markers located on the satellite region, we applied the fluorescent AFLP (amplified fragment length polymorphism) technique to aneuploids and deletion stocks of the cultivar T. aestivum 'Chinese Spring'. Out of 6017 fragments amplified with 80 primer combinations in normal 'Chinese Spring', 24 were assigned to 1BS. Twelve of them clustered within a small region of the satellite known to be rich in RFLP (restriction fragment length polymorphism) markers. AFLPs in 1BS and in the whole genome were calculated between 'Chinese Spring' and T. spelta var. duhamelianum. The polymorphism rates in the satellite region (58.3%) and in the 1BS arm (45.8%) were much higher than the average rate for the whole genome (10.7%). Seven of the 12 AFLP markers in the satellite region were revealed to be specific to 'Chinese Spring' and could potentially be useful for genetic mapping in a segregation population of 'Chinese Spring' × T. spelta.Key words: AFLP, wheat, deletion mapping, 1BS satellite.

scholarly journals PCR Fragment Length Polymorphism Analysis of Vancomycin-Resistant Enterococcus faecium

2000 ◽  
Vol 38 (8) ◽  
pp. 2885-2888 ◽  
Author(s):  
Susan Donabedian ◽  
Ellie Hershberger ◽  
Lee Ann Thal ◽  
J. W. Chow ◽  
Don B. Clewell ◽  
...  
Keyword(s):  
Length Polymorphism ◽  
Fragment Length ◽  
Enterococcus Faecium ◽  
Fragment Length Polymorphism ◽  
Pulsed Field Gel Electrophoresis ◽  
Polymorphism Analysis ◽  
Vancomycin Resistant Enterococcus ◽  
Glycopeptide Resistance ◽  
Pcr Product ◽  
Vancomycin Resistant

In this study, the glycopeptide resistance element, Tn1546, in 124 VanA Enterococcus faeciumclinical isolates from 13 Michigan hospitals was evaluated using PCR fragment length polymorphism. There were 26 pulsed-field gel electrophoresis (PFGE) types, which consisted of epidemiologically related and unrelated isolates from separate patients (1992 to 1996). Previously published oligonucleotides specific for regions in thevanA gene cluster of Tn1546 were used to amplify vanRS, vanSH, vanHAX,vanXY, and vanYZ. The glycopeptide resistance element, Tn1546, of E. faecium 228 was used as the basis of comparison for all the isolates in this study. Five PCR fragment length patterns were found, as follows. (i) PCR amplicons were the same size as those of EF228 for all genes in the vanAcluster in 19.4% of isolates. (ii) The PCR amplicon forvanSH was larger than that of EF228 (3.7 versus 2.3 kb) due to an insertion between the vanS and vanH genes (79.2% of isolates). (iii) One isolate in a unique PFGE group had avanSH amplicon larger than that of EF228 (5.7 versus 2.3 kb) due to an insertion in the vanS gene and an insertion between the vanS and vanH genes. (iv) One isolate did not produce a vanSH amplicon, but whenvanS and vanH were amplified separately, both amplicons were the same size as those as EF228. (v) One isolate had avanYZ PCR product larger than that of EF228 (2.8 versus 1.6 kb). This study shows that in a majority of the VanA E. faecium isolates, Tn1546 is altered compared to that of EF228. A total of 79.2% of the study isolates had the same-size insertion between the vanS and vanH genes. The results of this study show dissemination of an altered Tn1546 in heterologous VanA E. faecium in Michigan hospitals.

scholarly journals Evaluation of Fluorescence-Based Amplified Fragment Length Polymorphism Analysis for Molecular Typing in Hospital Epidemiology: Comparison with Pulsed-Field Gel Electrophoresis for Typing Strains of Vancomycin-Resistant Enterococcus faecium

2000 ◽  
Vol 38 (11) ◽  
pp. 4058-4065 ◽  
Author(s):  
Nick A. Antonishyn ◽  
Ryan R. McDonald ◽  
Edward L. Chan ◽  
Greg Horsman ◽  
Carla E. Woodmansee ◽  
...  
Keyword(s):  
Length Polymorphism ◽  
Fragment Length ◽  
Molecular Typing ◽  
Gel Electrophoresis ◽  
Fragment Length Polymorphism ◽  
Amplified Fragment Length Polymorphism ◽  
Pulsed Field Gel Electrophoresis ◽  
Restriction Fragments ◽  
Pulsed Field ◽  
Vancomycin Resistant

Fluorescence-based amplified fragment length polymorphism (fbAFLP) is a novel assay based on the fluorescent analysis of an amplified subset of restriction fragments. The fbAFLP assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The ligation of adapters with primer-specific sites coupled with primers containing selective nucleotides allowed the full potential of PCR to be realized while maintaining the advantages of restriction endonuclease analysis. Fluorescence-based fragment analysis with polyacrylamide gel electrophoresis provides the accurate band sizing required for homology assessment. The large number of phylogenetically informative characters obtained by fbAFLP is well suited for cluster analysis and database development. The method demonstrated excellent reproducibility and ease of performance and interpretation. We typed 30 epidemiologically well-characterized isolates of vancomycin-resistant enterococci from an outbreak in a university hospital by fbAFLP. Clustering of fbAFLP data matched epidemiological, microbiological, and pulsed-field gel electrophoresis data. This study demonstrates the unprecedented utility of fbAFLP for epidemiological investigation. Future developments in standardization and automation will set fbAFLP as the “gold standard” for molecular typing in epidemiology.

Sign in / Sign up

Export Citation Format

Share Document