Molecular Characterization of<i>Salmonella</i>Enteritidis: Comparison of an Optimized Multi-Locus Variable-Number of Tandem Repeat Analysis (MLVA) and Pulsed-Field Gel Electrophoresis

Dewaele, Isabelle; Rasschaert, Geertrui; Bertrand, Sophie; Wildemauwe, Christa; Wattiau, Pierre; Imberechts, Hein; Herman, Lieve; Ducatelle, Richard; Reu, Koen De; Heyndrickx, Marc

doi:10.1089/fpd.2012.1199

Cited by 18 publications

(16 citation statements)

References 36 publications

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“…Both MLVA and CRISPR-MVLST have been assessed in Salmonella based on these criteria (7,8,(10)(11)(12)(13). Evaluation of subtyping methods is often conducted through comparisons with PFGE; however, PFGE is not sufficiently discriminatory against clonal organisms such as S. enterica serotype Enteritidis and its utility as a benchmark for other subtyping techniques can be compromised.…”

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

et al. 2015

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A retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella enterica serotype Enteritidis and survey the population structure of commonly encountered S. enterica serotype Enteritidis outbreak isolates in the United States. A total of 52 S. enterica serotype 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 variablenumber 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 of S. enterica serotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods. Salmonella enterica is currently the most common bacterial foodborne pathogen in the United States, causing over 1 million cases of illnesses annually, including approximately 20,000 hospitalizations and 400 deaths (1). Serotyping is commonly used to subtype strains below the species level for epidemiologic purposes. Salmonella enterica serotype Enteritidis was the serotype most commonly linked to foodborne outbreaks between 1998 and 2008 in the United States, with shell eggs being the major vehicle for foodborne transmission (2). In recent years, S. enterica serotype Enteritidis was also found to cause multistate outbreaks associated with other foods such as ground beef (2012), Turkish pine nuts (2011), and alfalfa and spicy sprouts (2011), in addition to shelled eggs (2010) (3).During outbreak investigations, it is critical to employ subtyping methods capable of distinguishing outbreak isolates from epidemiologically distinct but genetically related bacterial strains. Most S. enterica serotype Enteritidis isolates have been shown to be genetically homogeneous, making it difficult for conventional subtyping methods such as pulsed-field gel electrophoresis (PFGE), the current gold standard for strain-level Salmonella subtyping, to discriminate between strains (4, 5). Among the S. enterica serotype Enteritidis isolates reported to PulseNet (6), approximately 45% display a single PFGE pattern using XbaI (JEGX01.0004), rendering PFGE ineffective in some foodborne outbreak investigations. One strategy to improve subtype resolution is to target hypervariable regions (i.e., regions of the bacterial chromosome with less genetic stability) in the ba...

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

et al. 2015

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“…Since repeat lengths in STTR3 can differ, this locus is sometimes expressed using allele codes as presented in reference 8 or as the total size in base pairs. Similar schemes are also in use or development for other S. enterica serovars, including Enteritidis, Gallinarum, Heidelberg, Dublin, and Typhi (12)(13)(14)(15)(16)(17)(18), and almost a hundred papers reported the use of MLVA for Salmonella analysis between 2004 and 2013 (see the supplemental material).…”

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Analysis of Salmonella enterica Serovar Typhimurium Variable-Number Tandem-Repeat Data for Public Health Investigation Based on Measured Mutation Rates and Whole-Genome Sequence Comparisons

et al. 2014

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c Variable-number tandem repeats (VNTRs) mutate rapidly and can be useful markers for genotyping. While multilocus VNTR analysis (MLVA) is increasingly used in the detection and investigation of food-borne outbreaks caused by Salmonella enterica serovar Typhimurium (S. Typhimurium) and other bacterial pathogens, MLVA data analysis usually relies on simple clustering approaches that may lead to incorrect interpretations. Here, we estimated the rates of copy number change at each of the five loci commonly used for S. Typhimurium MLVA, during in vitro and in vivo passage. We found that loci STTR5, STTR6, and STTR10 changed during passage but STTR3 and STTR9 did not. Relative rates of change were consistent across in vitro and in vivo growth and could be accurately estimated from diversity measures of natural variation observed during large outbreaks. Using a set of 203 isolates from a series of linked outbreaks and whole-genome sequencing of 12 representative isolates, we assessed the accuracy and utility of several alternative methods for analyzing and interpreting S. Typhimurium MLVA data. We show that eBURST analysis was accurate and informative. For construction of MLVA-based trees, a novel distance metric, based on the geometric model of VNTR evolution coupled with locus-specific weights, performed better than the commonly used simple or categorical distance metrics. The data suggest that, for the purpose of identifying potential transmission clusters for further investigation, isolates whose profiles differ at one of the rapidly changing STTR5, STTR6, and STTR10 loci should be collapsed into the same cluster.

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“…MLVA involves amplification and fragment size analysis of polymorphic regions of DNA that contain variable numbers of tandemly repeated sequences. This approach has been used most commonly for S. Typhimurium (6, 7), S. Enteritidis (8)(9)(10)(11), and S. Typhi (12). An additional study by van Cuyck and colleagues used MLVA to analyze 31 different Salmonella serovars (13).…”

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Subtyping of Salmonella enterica Serovar Newport Outbreak Isolates by CRISPR-MVLST and Determination of the Relationship between CRISPR-MVLST and PFGE Results

et al. 2013

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d Salmonella enterica subsp. enterica serovar Newport (S. Newport) is the third most prevalent cause of food-borne salmonellosis. Rapid, efficient, and accurate methods for identification are required to track specific strains of S. Newport during outbreaks. By exploiting the hypervariable nature of virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs), we previously developed a sequence-based subtyping approach, designated CRISPR-multi-virulence-locus sequence typing (CRISPR-MVLST). To demonstrate the applicability of this approach, we analyzed a broad set of S. Newport isolates collected over a 5-year period by using CRISPR-MVLST and pulsed-field gel electrophoresis (PFGE). Among 84 isolates, we defined 38 S. Newport sequence types (NSTs), all of which were novel compared to our previous analyses, and 62 different PFGE patterns. Our data suggest that both subtyping approaches have high discriminatory abilities (>0.95) with a potential for clustering cases with common exposures. Importantly, we found that isolates from closely related NSTs were often similar by PFGE profile as well, further corroborating the applicability of CRISPR-MVLST. In the first full application of CRISPR-MVLST, we analyzed isolates from a recent S. Newport outbreak. In this blinded study, we confirmed the utility of CRISPR-MVLST and were able to distinguish the 10 outbreak isolates, as defined by PFGE and epidemiological data, from a collection of 20 S. Newport isolates. Together, our data show that CRISPR-MVLST could be a complementary approach to PFGE subtyping for S. Newport.

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Molecular Characterization ofSalmonellaEnteritidis: Comparison of an Optimized Multi-Locus Variable-Number of Tandem Repeat Analysis (MLVA) and Pulsed-Field Gel Electrophoresis

Cited by 18 publications

References 36 publications

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

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

Analysis of Salmonella enterica Serovar Typhimurium Variable-Number Tandem-Repeat Data for Public Health Investigation Based on Measured Mutation Rates and Whole-Genome Sequence Comparisons

Subtyping of Salmonella enterica Serovar Newport Outbreak Isolates by CRISPR-MVLST and Determination of the Relationship between CRISPR-MVLST and PFGE Results

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