CRISPR 2 PCR and high resolution melting profiling for identification and characterization of clinically-relevant Salmonella enterica subsp. enterica

Nuttachat Wisittipanit,Rungthiwa Srimora,Nattinee Kittiwan,Kritchai Poonchareon,Chaiwat Pulsrikarn,Sudarat Srisong

doi:10.7717/peerj.9113

Nuttachat Wisittipanit, Rungthiwa Srimora + Show 4 more

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https://doi.org/10.7717/peerj.9113

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Abstract

BackgroundNontyphoidal Salmonella spp. constitute a major bacterial cause of food poisoning. Each Salmonella serotype causes distinct virulence to humans.MethodA small cohort study was conducted to characterize several aspects of Salmonella isolates obtained from stool of diarrheal patients (n = 26) admitted to Phayao Ram Hospital, Phayao province, Thailand. A simple CRISPR 2 molecular analysis was developed to rapidly type Salmonella isolates employing both uniplex and high resolution melting (HRM) curve analysis.ResultsCRISPR 2 monoplex PCR generated a single Salmonella serotype-specific amplicon, showing S. 4,[5],12:i:- with highest frequency (42%), S. Enteritidis (15%) and S. Stanley (11%); S. Typhimurium was not detected. CRISPR 2 HRM-PCR allowed further classification of S. 4,[5],12:i:- isolates based on their specific CRISPR 2 signature sequences. The highest prevalence of Salmonella infection was during the summer season (April to August). Additional studies were conducted using standard multiplex HRM-PCR typing, which confirmed CRISPR 2 PCR results and, using a machine-learning algorithm, clustered the majority of Salmonella serotypes into six clades; repetitive element-based (ERIC) PCR, which clustered the serotypes into three clades only; antibiogram profiling, which revealed the majority resistant to ampicillin (69%); and test for extended spectrum β-lactamase production (two isolates) and PCR-based detection of bla alleles.ConclusionCRISPR 2 PCR provided a simple assay for detection and identification of clinically-relevant Salmonella serotypes. In conjunction with antibiogram profiling and rapid assay for β-lactamase producers, this approach should facilitate detection and appropriate treatment of Salmonellosis in a local hospital setting. In addition, CRISPR 2 HRM-PCR profiling enabled clustering of S. 4,[5],12:i:-isolates according to CRISPR 2 locus signature sequences, indicative of their different evolutionary trajectories, thereby providing a powerful tool for future epidemiological studies of virulent Salmonella serotypes.

Highlights

Salmonella enterica can cause human gastroenteritis owing to inadequate hygienic standards of living and/or consuming poorly prepared fresh food
Samples were incubated in buffered peptone water (Oxoid, Hampshire, UK) at 37 ◦C overnight, plated on SS and XLD agar (Oxoid, Hampshire, UK), and suspected Salmonella colonies indicated by using a triple sugar iron (TSI) slant (Oxoid, Hampshire, UK) assay and lysine iron agar (LIA) (Biomedia, Nontanuri, Thailand)
Multiplex high resolution melting (HRM)-PCR assay of Salmonella serotypes, not able to type all eight serotypes responsible for food poisoning, is adequate in identifying the majority of pertinent clinical Salmonella serotypes in particular S. 4,[5],12:i:- and S

Summary

Introduction

Salmonella enterica can cause human gastroenteritis owing to inadequate hygienic standards of living and/or consuming poorly prepared fresh food. Each Salmonella serotype causes distinct virulence to humans. CRISPR 2 monoplex PCR generated a single Salmonella serotype-specific amplicon, showing S. Additional studies were conducted using standard multiplex HRM-PCR typing, which confirmed CRISPR 2 PCR results and, using a machinelearning algorithm, clustered the majority of Salmonella serotypes into six clades; repetitive element-based (ERIC) PCR, which clustered the serotypes into three clades only; antibiogram profiling, which revealed the majority resistant to ampicillin (69%); and test for extended spectrum β-lactamase production (two isolates) and PCR-based detection of bla alleles. CRISPR 2 PCR provided a simple assay for detection and identification of clinically-relevant Salmonella serotypes. 4,[5],12:i:-isolates according to CRISPR 2 locus signature sequences, indicative of their different evolutionary trajectories, thereby providing a powerful tool for future epidemiological studies of virulent Salmonella serotypes CRISPR 2 HRM-PCR profiling enabled clustering of S. 4,[5],12:i:-isolates according to CRISPR 2 locus signature sequences, indicative of their different evolutionary trajectories, thereby providing a powerful tool for future epidemiological studies of virulent Salmonella serotypes

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