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

Wang, Qinning; Kong, Fanrong; Jelfs, Peter; Gilbert, Gwendolyn L.

doi:10.1099/jmm.0.47766-0

Cited by 16 publications

(18 citation statements)

References 20 publications

(27 reference statements)

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“…We performed sPCRs, using the same primers as in the mPCR, and uniformly yielded amplicons of the expected size for all VF genes with mPCR/RLB probe-pair discrepancies, suggesting that withinprobe-pair discordance resulted from sequence variation within the target region of the signal-negative probes. This was confirmed by sequencing of selected sPCR products (data not shown) and supports previous work by others (18,19,23,26,32).…”

Section: Discussionsupporting

confidence: 75%

Multiplex PCR-Based Reverse Line Blot Assay for Simultaneous Detection of 22 Virulence Genes in Uropathogenic Escherichia coli

Kudinha

Kong

Johnson

et al. 2012

Appl Environ Microbiol

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Urinary tract infections (UTIs) are among the most common bacterial infections and are responsible for significant morbidity and health care costs worldwide. The main bacterial cause of uncomplicated UTI is Escherichia coli, which possesses numerous virulence factors (VFs). Many studies of the pathogenesis of E. coli UTI have centered on VF genes. Hence, the development of better molecular assays to study VF genes would facilitate these studies. We developed a highly sensitive and specific multiplex PCR-based reverse line blot (mPCR/RLB) assay to simultaneously detect 22 VF genes of uropathogenic E. coli and then used it to characterize 180 isolates from nonpregnant women of child-bearing age with cystitis and 153 fecal isolates from similar-age healthy women, in regional New South Wales, Australia. The assay accurately identified all VF genes (of the 22 under study) known to be present in 30 previously characterized control strains. The detection limits were 28 ng of DNA from E. coli isolates and 50 CFU/ml in mock-infected urine specimens containing known concentrations of E. coli. Cystitis isolates (compared to the fecal isolates) showed a significantly higher prevalence of 18 individual VF genes and contained significantly more VF genes per isolate (median number, 18.5 versus 6.5 [P ‫؍‬ 0.001]). Discordance between paired probes for a given VF gene occurred in several clinical test isolates but no reference strains and among the test isolates was associated with fecal source (10% of VF genes versus 2% for cystitis isolates [P < 0.001]). This novel mPCR/RLB method is a potentially powerful tool for investigating the prevalence and distribution of VFs in E. coli.

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Section: Discussionsupporting

confidence: 75%

Multiplex PCR-Based Reverse Line Blot Assay for Simultaneous Detection of 22 Virulence Genes in Uropathogenic Escherichia coli

Kudinha

Kong

Johnson

et al. 2012

Appl Environ Microbiol

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“…Cluster analysis revealed that isolates with the same SCCmec subtype, as defined by Milheiriço et al (21), did not always group together and that none of the 52 reference strains corresponded exactly with the six type strains, suggesting that existing SCCmec type IV subtyping methods may not be able to represent complex phylogenetic relationships for this diverse MRSA type. Weak signals were noted for some probes with a few isolates, which were likely due to minor sequence variations in the probe regions, as reported previously (28).…”

Section: Resultssupporting

confidence: 55%

Unexpected Diversity of Staphylococcal Cassette Chromosome mec Type IV in Methicillin-Resistant Staphylococcus aureus Strains

et al. 2010

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Staphylococcal cassette chromosome mec (SCCmec) is a large mobile genetic element which is used frequently for subtyping of methicillin-resistant Staphylococcus aureus (MRSA) strains. MRSA SCCmec type IV not only predominates among community-acquired MRSA (CA-MRSA) strains but also is associated with several genetic lineages of hospital-acquired MRSA (HA-MRSA) and with other species. The objective of this study was to investigate the diversity of MRSA strains classified as SCCmec type IV by using a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay as well as spa typing and pulsed-field gel electrophoresis (PFGE). Sixty-two primer pairs and 63 probes were designed to interrogate each open reading frame (ORF) of SCCmec type IV sequences. A set of 131 MRSA SCCmec type IV isolates were classified into 79 subtypes by this method. There was considerable concordance between SCCmec type IV subtyping, spa typing, and PFGE patterns for clinical isolates, and the stability of SCCmec type IV subtyping was comparable to that of the other two methods. Using an in-house computer program, we showed that a subset of 20 genetic markers could achieve the same level of discrimination between isolates as the full set of 62, with a Simpson's index of diversity of 0.975. SCCmec type IV has a much higher level of diversity than previously suggested. The application of the mPCR/RLB hybridization assay to MRSA SCCmec type IV subtyping can improve the discriminatory power and throughput of MRSA typing and has the potential to enhance rapid infection control surveillance and outbreak detection.

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“…Outbreak 1 occurred in a residential college in November 2006, when 16 cases of severe gastroenteritis were diagnosed among students and staff over 2 days (23). S. Typhimurium DT170 with MLVA profile 3-11-7-12-523 was recovered from stool samples from all of the cases.…”

Section: Selection Of Isolates and Genomic Variations Between Smentioning

confidence: 99%

Delineating Community Outbreaks of Salmonella enterica Serovar Typhimurium by Use of Whole-Genome Sequencing: Insights into Genomic Variability within an Outbreak

et al. 2015

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c Whole-genome next-generation sequencing (NGS) was used to retrospectively examine 57 isolates from five epidemiologically confirmed community outbreaks (numbered 1 to 5) caused by Salmonella enterica serovar Typhimurium phage type DT170. Most of the human and environmental isolates confirmed epidemiologically to be involved in the outbreaks were either genomically identical or differed by one or two single nucleotide polymorphisms (SNPs), with the exception of those in outbreak 1. The isolates from outbreak 1 differed by up to 12 SNPs, which suggests that the food source of the outbreak was contaminated with more than one strain while each of the other four outbreaks was caused by a single strain. In addition, NGS analysis ruled in isolates that were initially not considered to be linked with the outbreak, which increased the total outbreak size by 107%. The mutation process was modeled by using known mutation rates to derive a cutoff value for the number of SNP difference to determine whether or not a case was part of an outbreak. For an outbreak with less than 1 month of ex vivo/in vivo evolution time, the maximum number of SNP differences between isolates is two or four using the lowest or highest mutation rate, respectively. NGS of S. Typhimurium significantly increases the resolution of investigations of community outbreaks. It can also inform a more targeted public health response by providing important supplementary evidence that cases of disease are or are not associated with food-borne outbreaks of S. Typhimurium. Salmonella enterica serovar Typhimurium is the most common serovar isolated from humans and animals in Australia. Traditionally, surveillance and outbreak investigations of S. Typhimurium rely upon phage typing, which is based on the susceptibility of isolates to a set of bacteriophages. Phage type DT170 has been increasing steadily over the last decade in Australia and became the most frequent phage type in 2004. Therefore, phage typing has limited resolution for outbreak detection. More recently, multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) has been adopted in public health reference laboratories across Australia for epidemiological typing because of its relatively high discriminatory power and ability to be harmonized (1). MLVA has also been used as a standardized method for outbreak detection in Europe (2, 3).In New South Wales (NSW), Australia, all Salmonella isolates from public and private pathology providers are routinely referred to the NSW Enteric Reference Laboratory, Institute for Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, for serotyping and MLVA typing using five VNTR loci (MLVA-5) (1). Prospective MLVA typing of S. Typhimurium has been useful for identifying outbreak clusters (1). In our current practice, recovery of five or more geographically clustered isolates of the same MLVA profile from patients with diarrhea within a 4-week period (cases in the same household are counted as one episode) signals that an outbreak has occurred and...

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

Cited by 16 publications

References 20 publications

Multiplex PCR-Based Reverse Line Blot Assay for Simultaneous Detection of 22 Virulence Genes in Uropathogenic Escherichia coli

Multiplex PCR-Based Reverse Line Blot Assay for Simultaneous Detection of 22 Virulence Genes in Uropathogenic Escherichia coli

Unexpected Diversity of Staphylococcal Cassette Chromosome mec Type IV in Methicillin-Resistant Staphylococcus aureus Strains

Delineating Community Outbreaks of Salmonella enterica Serovar Typhimurium by Use of Whole-Genome Sequencing: Insights into Genomic Variability within an Outbreak

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