scholarly journals The utility of multiple molecular methods including whole genome sequencing as tools to differentiate Escherichia coli O157:H7 outbreaks

2015 ◽  
Vol 20 (47) ◽  
Author(s):  
Byron M Berenger ◽  
Chrystal Berry ◽  
Trevor Peterson ◽  
Patrick Fach ◽  
Sabine Delannoy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Core Genome ◽  
Virulence Genes ◽  
Variable Number Tandem Repeat ◽  
Virulence Gene ◽  
Variable Number ◽  
Gene Profiling ◽  
Whole Genome

A standardised method for determining Escherichia coli O157:H7 strain relatedness using whole genome sequencing or virulence gene profiling is not yet established. We sought to assess the capacity of either high-throughput polymerase chain reaction (PCR) of 49 virulence genes, core-genome single nt variants (SNVs) or k-mer clustering to discriminate between outbreak-associated and sporadic E. coli O157:H7 isolates. Three outbreaks and multiple sporadic isolates from the province of Alberta, Canada were included in the study. Two of the outbreaks occurred concurrently in 2014 and one occurred in 2012. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem repeat analysis (MLVA) were employed as comparator typing methods. The virulence gene profiles of isolates from the 2012 and 2014 Alberta outbreak events and contemporary sporadic isolates were mostly identical; therefore the set of virulence genes chosen in this study were not discriminatory enough to distinguish between outbreak clusters. Concordant with PFGE and MLVA results, core genome SNV and k-mer phylogenies clustered isolates from the 2012 and 2014 outbreaks as distinct events. k-mer phylogenies demonstrated increased discriminatory power compared with core SNV phylogenies. Prior to the widespread implementation of whole genome sequencing for routine public health use, issues surrounding cost, technical expertise, software standardisation, and data sharing/comparisons must be addressed.

scholarly journals Impact of whole genome sequencing on the investigation of food-borne outbreaks of Shiga toxin-producing Escherichia coli serogroup O157:H7, England, 2013 to 2017

2019 ◽  
Vol 24 (4) ◽  
Author(s):  
Claire Jenkins ◽  
Timothy J Dallman ◽  
Kathie A Grant
Keyword(s):  
Escherichia Coli ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Shiga Toxin ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Whole Genome ◽  
Trace Back ◽  
Food Borne ◽  
Evolutionary Context

We aim to provide insight and guidance on the utility of whole genome sequencing (WGS) data for investigating food-borne outbreaks of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in England between 2013 and 2017. Analysis of WGS data delivered an unprecedented level of strain discrimination when compared with multilocus variable number tandem repeat analysis. The robustness of the WGS method ensured confidence in the microbiological identification of linked cases, even when epidemiological links were obscured. There was evidence that phylogeny derived from WGS data can be used to trace the geographical origin of an isolate. Further analysis of the phylogenetic data provided insight on the evolutionary context of emerging pathogenic strains. Publically available WGS data linked to the clinical, epidemiological and environmental context of the sequenced strain has improved trace back investigations during outbreaks. Expanding the use of WGS-based typing analysis globally will ensure the rapid implementation of interventions to protect public health, inform risk assessment and facilitate the management of national and international food-borne outbreaks of STEC O157:H7.

scholarly journals Genetic Characterization of Brucella spp.: Whole Genome Sequencing-Based Approach for the Determination of Multiple Locus Variable Number Tandem Repeat Profiles

2021 ◽  
Vol 12 ◽  
Author(s):  
Ana Pelerito ◽  
Alexandra Nunes ◽  
Teresa Grilo ◽  
Joana Isidro ◽  
Catarina Silva ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
In Silico ◽  
Variable Number Tandem Repeat ◽  
Epidemiological Studies ◽  
Variable Number ◽  
Epidemiological Surveillance ◽  
Future Application ◽  
Whole Genome

Brucellosis is an important zoonosis that is emerging in some regions of the world, gaining increased relevance with the inclusion of the causing agent Brucella spp. in the class B bioterrorism group. Until now, multi-locus VNTR Analysis (MLVA) based on 16 loci has been considered as the gold standard for Brucella typing. However, this methodology is laborious, and, with the rampant release of Brucella genomes, the transition from the traditional MLVA to whole genome sequencing (WGS)-based typing is on course. Nevertheless, in order to avoid a disruptive transition with the loss of massive genetic data obtained throughout the last decade and considering that the transition timings will vary considerably among different countries, it is important to determine WGS-based MLVA alleles of the nowadays sequenced genomes. On this regard, we aimed to evaluate the performance of a Python script that had been previously developed for the rapid in silico extraction of the MLVA alleles, by comparing it to the PCR-based MLVA procedure over 83 strains from different Brucella species. The WGS-based MLVA approach detected 95.3% of all possible 1,328 hits (83 strains×16 loci) and showed an agreement rate with the PCR-based MLVA procedure of 96.4% for MLVA-16. According to our dataset, we suggest the use of a minimal depth of coverage of ~50x and a maximum number of ~200 contigs as guiding “boundaries” for the future application of the script. In conclusion, the evaluated script seems to be a very useful and robust tool for the in silico determination of MLVA profiles of Brucella strains, allowing retrospective and prospective molecular epidemiological studies, which are important for maintaining an active epidemiological surveillance of brucellosis.

scholarly journals Genome-based characterization of Escherichia coli causing bloodstream infection through next-generation sequencing

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244358
Author(s):  
Rafika Indah Paramita ◽  
Erni Juwita Nelwan ◽  
Fadilah Fadilah ◽  
Editha Renesteen ◽  
Nelly Puspandari ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Whole Genome Sequencing ◽  
Bloodstream Infection ◽  
Genome Sequencing ◽  
Virulence Genes ◽  
Sequence Type ◽  
Whole Genome ◽  
National Hospital ◽  
Sequence Types

Escherichia coli are one of the commonest bacteria causing bloodstream infection (BSI). The aim of the research was to identify the serotypes, MLST (Multi Locus Sequence Type), virulence genes, and antimicrobial resistance of E. coli isolated from bloodstream infection hospitalized patients in Cipto Mangunkusumo National Hospital Jakarta. We used whole genome sequencing methods rather than the conventional one, to characterized the serotypes, MLST (Multi Locus Sequence Type), virulence genes, and antimicrobial resistance (AMR) of E. coli. The composition of E. coli sequence types (ST) was as follows: ST131 (n = 5), ST38 (n = 3), ST405 (n = 3), ST69 (n = 3), and other STs (ST1057, ST127, ST167, ST3033, ST349, ST40, ST58, ST6630). Enteroaggregative E. coli (EAEC) and Extra-intestinal pathogenic E. coli (ExPEC) groups were found dominant in our samples. Twenty isolates carried virulence genes for host cells adherence and 15 for genes that encourage E. coli immune evasion by enhancing survival in serum. ESBL-genes were present in 17 E. coli isolates. Other AMR genes also encoded resistance against aminoglycosides, quinolones, chloramphenicol, macrolides and trimethoprim. The phylogeny analysis showed that phylogroup D is dominated and followed by phylogroup B2. The E. coli isolated from 22 patients in Cipto Mangunkusumo National Hospital Jakarta showed high diversity in serotypes, sequence types, virulence genes, and AMR genes. Based on this finding, routinely screening all bacterial isolates in health care facilities can improve clinical significance. By using Whole Genome Sequencing for laboratory-based surveillance can be a valuable early warning system for emerging pathogens and resistance mechanisms.

scholarly journals Whole Genome Sequencing Characterization of Shiga Toxin–Producing Escherichia coli Isolated from Flour from Swiss Retail Markets

2019 ◽  
Vol 82 (8) ◽  
pp. 1398-1404 ◽  
Author(s):  
RENATE BOSS ◽  
JOERG HUMMERJOHANN
Keyword(s):  
Escherichia Coli ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Shiga Toxin ◽  
Severe Disease ◽  
Virulence Gene ◽  
Whole Genome ◽  
N 93 ◽  
Human Infections

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) strains are often found in food and cause human infections. Although STEC O157:H7 is most often responsible for human disease, various non-O157 subtypes have caused individual human infections or outbreaks. The importance of STEC serogroup typing is decreasing while detection of virulence gene patterns has become more relevant. Whole genome sequencing (WGS) reveals the entire spectrum of pathogen information, such as toxin variant, serotype, sequence type, and virulence factors. Flour has not been considered as a vector for STEC; however, this product has been associated with several STEC outbreaks in the last decade. Flour is a natural product, and milling does not include a germ-reducing step. Flour is rarely eaten raw, but the risks associated with the consumption of unbaked dough are probably underestimated. The aim of this study was to determine the prevalence of STEC in flour samples (n = 93) collected from Swiss markets and to fully characterize the isolates by PCR assay and WGS. The prevalence of STEC in these flour samples was 10.8% as indicated by PCR, and a total of 10 STEC strains were isolated (two flour samples were positive for two STEC subtypes). We found one stx2-positve STEC isolate belonging to the classic serogroups frequently associated with outbreaks that could potentially cause severe disease. However, we also found several other common or less common STEC subtypes with diverse virulence patterns. Our results reveal the benefits of WGS as a characterization tool and that flour is a potentially and probably underestimated source for STEC infections in humans.

scholarly journals Prospective Whole-Genome Sequencing Enhances National Surveillance of Listeria monocytogenes

2015 ◽  
Vol 54 (2) ◽  
pp. 333-342 ◽  
Author(s):  
Jason C. Kwong ◽  
Karolina Mercoulia ◽  
Takehiro Tomita ◽  
Marion Easton ◽  
Hua Y. Li ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Genetic Population ◽  
Content Type ◽  
Epidemiologic Surveillance

Whole-genome sequencing (WGS) has emerged as a powerful tool for comparing bacterial isolates in outbreak detection and investigation. Here we demonstrate that WGS performed prospectively for national epidemiologic surveillance ofListeria monocytogeneshas the capacity to be superior to our current approaches using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA), binary typing, and serotyping. Initially 423L. monocytogenesisolates underwent WGS, and comparisons uncovered a diverse genetic population structure derived from three distinct lineages. MLST, binary typing, and serotyping results inferredin silicofrom the WGS data were highly concordant (>99%) with laboratory typing performed in parallel. However, WGS was able to identify distinct nested clusters within groups of isolates that were otherwise indistinguishable using our current typing methods. Routine WGS was then used for prospective epidemiologic surveillance on a further 97L. monocytogenesisolates over a 12-month period, which provided a greater level of discrimination than that of conventional typing for inferring linkage to point source outbreaks. A risk-based alert system based on WGS similarity was used to inform epidemiologists required to act on the data. Our experience shows that WGS can be adopted for prospectiveL. monocytogenessurveillance and investigated for other pathogens relevant to public health.

scholarly journals Navigating Microbiological Food Safety in the Era of Whole-Genome Sequencing

2016 ◽  
Vol 29 (4) ◽  
pp. 837-857 ◽  
Author(s):  
J. Ronholm ◽  
Neda Nasheri ◽  
Nicholas Petronella ◽  
Franco Pagotto
Keyword(s):  
Food Safety ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Antibiotic Resistance Gene ◽  
Virulence Gene ◽  
Complete Information ◽  
Gene Profiling ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Molecular Subtyping

SUMMARYThe epidemiological investigation of a foodborne outbreak, including identification of related cases, source attribution, and development of intervention strategies, relies heavily on the ability to subtype the etiological agent at a high enough resolution to differentiate related from nonrelated cases. Historically, several different molecular subtyping methods have been used for this purpose; however, emerging techniques, such as single nucleotide polymorphism (SNP)-based techniques, that use whole-genome sequencing (WGS) offer a resolution that was previously not possible. With WGS, unlike traditional subtyping methods that lack complete information, data can be used to elucidate phylogenetic relationships and disease-causing lineages can be tracked and monitored over time. The subtyping resolution and evolutionary context provided by WGS data allow investigators to connect related illnesses that would be missed by traditional techniques. The added advantage of data generated by WGS is that these data can also be used for secondary analyses, such as virulence gene detection, antibiotic resistance gene profiling, synteny comparisons, mobile genetic element identification, and geographic attribution. In addition, several software packages are now available to generatein silicoresults for traditional molecular subtyping methods from the whole-genome sequence, allowing for efficient comparison with historical databases. Metagenomic approaches using next-generation sequencing have also been successful in the detection of nonculturable foodborne pathogens. This review addresses state-of-the-art techniques in microbial WGS and analysis and then discusses how this technology can be used to help support food safety investigations. Retrospective outbreak investigations using WGS are presented to provide organism-specific examples of the benefits, and challenges, associated with WGS in comparison to traditional molecular subtyping techniques.

scholarly journals Comparative Analysis of Subtyping Methods against a Whole-Genome-Sequencing Standard for Salmonella enterica Serotype Enteritidis

2014 ◽  
Vol 53 (1) ◽  
pp. 212-218 ◽  
Author(s):  
Xiangyu Deng ◽  
Nikki Shariat ◽  
Elizabeth M. Driebe ◽  
Chandler C. Roe ◽  
Beth Tolar ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Salmonella Enterica ◽  
Variable Number Tandem Repeat ◽  
The United States ◽  
Variable Number ◽  
Whole Genome ◽  
Molecular Subtyping ◽  
Content Type ◽  
Sporadic Cases

A retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods forSalmonella entericaserotype Enteritidis and survey the population structure of commonly encounteredS. entericaserotype Enteritidis outbreak isolates in the United States. A total of 52S. entericaserotype Enteritidis isolates representing 16 major outbreaks and three sporadic cases collected between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: (i) whole-genome single-nucleotide-polymorphism typing (WGST), (ii) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), (iii) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST), and (iv) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference results with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed the other methods in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins ofS. entericaserotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods.

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