Multiple-locus variable-number tandem repeat analysis of<i>Salmonella</i>Enteritidis isolates from human and non-human sources using a single multiplex PCR

Cho, Seongbeom; Boxrud, David; Bartkus, Joanne M.; Whittam, Thomas S.; Saeed, Mahdi

doi:10.1111/j.1574-6968.2007.00875.x

Cited by 46 publications

(49 citation statements)

References 32 publications

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“…Because of its greater discriminatory capacity, MLVA was used to assess the genetic similarity among S. Enteritidis isolates tested in this study Cho et al, 2007;Malorny et al, 2008). The majority (90 %) of isolates were grouped into eight clusters (clusters 4, 5 and 8-13) with a withincluster allelic congruence of ¢85 % (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…A chromosomally located sdfI gene reported to be unique to the serovar S. Enteritidis was targeted for the serotype-specific identification of S. Enteritidis (Agron et al, 2001;Alvarez et al, 2004;Clavijo et al, 2006;Trafny et al, 2006). A 60 kb virulence-plasmid-associated spvB gene was used as a marker for determination of the presence or absence of a typical virulence plasmid (pSEV) among S. Enteritidis isolates (Cho et al, 2007;Rotger & Casadesú s, 1999;Rychlík et al, 2006Rychlík et al, , 2008. PCR amplification of complete ORFs of the sipA, sipD, flgK, fljB and flgL genes was performed to detect the presence or absence of these genes among selected S. Enteritidis isolates.…”

Section: Methodsmentioning

confidence: 99%

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Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

et al. 2011

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Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne gastroenteritis in humans worldwide. Poultry and poultry products are considered the major vehicles of transmission to humans. Using cell invasiveness as a surrogate marker for pathogenicity, we tested the invasiveness of 53 poultry-associated isolates of S. Enteritidis in a well-differentiated intestinal epithelial cell model (Caco-2). The method allowed classification of the isolates into low (n57), medium (n518) and high (n530) invasiveness categories. Cell invasiveness of the isolates did not correlate with the presence of the virulence-associated gene spvB or the ability of the isolates to form biofilms. Testing of representative isolates with high and low invasiveness in a mouse model revealed that the former were more invasive in vivo and caused more and earlier mortalities, whereas the latter were significantly less invasive in vivo, causing few or no mortalities. Further characterization of representative isolates with low and high invasiveness showed that most of the isolates with low invasiveness had impaired motility and impaired secretion of either flagella-associated proteins (FlgK, FljB and FlgL) or type III secretion system (TTSS)-secreted proteins (SipA and SipD) encoded on Salmonella pathogenicity island-1. In addition, isolates with low invasiveness had impaired ability to invade and/or survive within chicken macrophages. These data suggest that not all isolates of S. Enteritidis recovered from poultry may be equally pathogenic, and that the pathogenicity of S. Enteritidis isolates is associated, in part, with both motility and secretion of TTSS effector proteins. INTRODUCTIONSalmonella enterica serovar Enteritidis (S. Enteritidis) is the leading cause of food-borne salmonellosis (WHO, 2008). S. Enteritidis-induced salmonellosis in humans is characterized by diarrhoea, fever, headache, abdominal pain, nausea and vomiting (CDC, 2007). S. Enteritidis is also increasingly reported from cases of invasive and extra-intestinal infections such as septicaemia, arthritis, endocarditis, meningitis and urinary tract infections (Ghosh & Vogt, 2006; Gordon et al., 2008;Katsenos et al., 2008; Kobayashi et al., 2009;Morpeth et al., 2009;Mutlu et al., 2009;Tena et al., 2007). Between 1990 and 2001, the US state and territorial health departments reported 677 S. Enteritidis outbreaks, which accounted for 23 366 illnesses, 1988 hospitalizations and 33 deaths (CDC, 2003). In 2006, countries within the European Union reported 1729 outbreaks caused by S. Enteritidis leading to 13 853 illnesses, 2714 hospitalizations and 14 deaths (EFSA, 2007). The Health Protection Agency of the UK reported 4194 cases of foodborne S. Enteritidis infection in (HPA, 2008. Poultry is considered the single largest reservoir of S. Enteritidis and most risk attribution studies have identified poultry and poultry products as the major source of human infection. S. Enteritidis is passed to humans mainly via handling and consumption of contaminated po...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

et al. 2011

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“…Some are relatively difficult to perform, slow and expensive whilst others have limited reproducibility and discriminatory powers. Multilocus variable-number tandem-repeat analysis (MLVA) has been developed for various Salmonella serovars (Cho et al, 2007;Lindstedt et al, 2004;Liu et al, 2003;Witonski et al, 2006). It has allowed a high level of resolution of variant strains of the relatively homogeneous S. Typhimurium definitive type 104 (Lindstedt et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Extended phage locus typing of Salmonella enterica serovar Typhimurium, using multiplex PCR-based reverse line blot hybridization

Wang

Kong

Jelfs

et al. 2008

Journal of Medical Microbiology

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Salmonella enterica serovar Typhimurium (S. Typhimurium) is the commonest pathogen causing food-borne disease among humans and animals in Australia. A multiplex PCR-based reverse line blot (mPCR/RLB) system was developed to rapidly identify S. Typhimurium phage types and strains within them. The system comprised 32 biotin-labelled primer sets and 38 amino-labelled probes, based on sequences that were either phage-type-related or derived from temperate phages ST64B, P22, Gifsy-1 or Gifsy-2. The system was developed and evaluated using 168 S. Typhimurium isolates, representing 46 phage types. RLB patterns, based on a combination of positive hybridization and grading of signal intensities, validated by sequencing, differentiated S. Typhimurium isolates into 102 types. Some clusters contained isolates belonging to a single phage type while others contained isolates belonging to more than one. Most phage types exhibited at least two RLB profiles. The feasibility of this system was evaluated during investigations of three outbreaks, due to two different phage types. Within each outbreak, isolates showed identical RLB patterns, whereas sporadic isolates of corresponding phage types showed various patterns. The mPCR/RLB system was compared with multilocus variable-number tandem-repeat analysis (MLVA). The two methods demonstrated similar discriminatory abilities. Based on these preliminary results, the mPCR/RLB system is a promising tool for molecular identification of most common S. Typhimurium phage types. It could be used as an alternative to, or in conjunction with, MLVA for rapid strain typing during outbreaks.

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“…Multilocus variable-number tandem-repeat analysis (MLVA) utilizes the polymorphism in the copy numbers of tandemly repeated sequences at multiple loci in the S. enterica serotype Enteritidis genome. It provides higher resolution than PFGE (7,8) and has become a supplementary subtyping technique for surveillance and investigation of S. enterica serotype Enteritidis outbreaks by PulseNet. Analysis using clustered regularly interspaced short palindromic repeats (CRISPRs) combined with multi-virulence-locus sequence typing (designated CRISPR-MVLST) takes advantage of combined sequence variations in the spacer regions of the two CRISPR loci in Salmonella and two virulence genes (fimH and sseL) (9).…”

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confidence: 99%

“…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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confidence: 99%

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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Multiple-locus variable-number tandem repeat analysis ofSalmonellaEnteritidis isolates from human and non-human sources using a single multiplex PCR

Cited by 46 publications

References 32 publications

Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

Extended phage locus typing of Salmonella enterica serovar Typhimurium, using multiplex PCR-based reverse line blot hybridization

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

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