Dataset Information

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

ABSTRACT:

Background

Nontyphoidal Salmonella spp. constitute a major bacterial cause of food poisoning. Each Salmonella serotype causes distinct virulence to humans.

Method

A 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.

Results

CRISPR 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 ?-lactamase production (two isolates) and PCR-based detection of bla alleles.

Conclusion

CRISPR 2 PCR provided a simple assay for detection and identification of clinically-relevant Salmonella serotypes. In conjunction with antibiogram profiling and rapid assay for ?-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.

SUBMITTER: Wisittipanit N

PROVIDER: S-EPMC7304428 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Wisittipanit Nuttachat N Pulsrikarn Chaiwat C Srisong Sudarat S Srimora Rungthiwa R Kittiwan Nattinee N Poonchareon Kritchai K

PeerJ 20200616

<h4>Background</h4>Nontyphoidal <i>Salmonella</i> spp. constitute a major bacterial cause of food poisoning. Each <i>Salmonella</i> serotype causes distinct virulence to humans.<h4>Method</h4>A small cohort study was conducted to characterize several aspects of <i>Salmonella</i> isolates obtained from stool of diarrheal patients (<i>n</i> = 26) admitted to Phayao Ram Hospital, Phayao province, Thailand. A simple CRISPR 2 molecular analysis was developed to rapidly type <i>Salmonella</i> isolates ...[more]

PMID: 32587791

Similar Datasets

Project description:BACKGROUND:Bacteriophages are bacterial parasites and are considered the most abundant and diverse biological entities on the planet. Previously we identified 154 prophages from 151 serovars of Salmonella enterica subsp. enterica. A detailed analysis of Salmonella prophage genomics is required given the influence of phages on their bacterial hosts and should provide a broader understanding of Salmonella biology and virulence and contribute to the practical applications of phages as vectors and antibacterial agents. RESULTS:Here we provide a comparative analysis of the full genome sequences of 142 prophages of Salmonella enterica subsp. enterica which is the full complement of the prophages that could be retrieved from public databases. We discovered extensive variation in genome sizes (ranging from 6.4 to 358.7?kb) and guanine plus cytosine (GC) content (ranging from 35.5 to 65.4%) and observed a linear correlation between the genome size and the number of open reading frames (ORFs). We used three approaches to compare the phage genomes. The NUCmer/MUMmer genome alignment tool was used to evaluate linkages and correlations based on nucleotide identity between genomes. Multiple sequence alignment was performed to calculate genome average nucleotide identity using the Kalgin program. Finally, genome synteny was explored using dot plot analysis. We found that 90 phage genome sequences grouped into 17 distinct clusters while the remaining 52 genomes showed no close relationships with the other phage genomes and are identified as singletons. We generated genome maps using nucleotide and amino acid sequences which allowed protein-coding genes to be sorted into phamilies (phams) using the Phamerator software. Out of 5796 total assigned phamilies, one phamily was observed to be dominant and was found in 49 prophages, or 34.5% of the 142 phages in our collection. A majority of the phamilies, 4330 out of 5796 (74.7%), occurred in just one prophage underscoring the high degree of diversity among Salmonella bacteriophages. CONCLUSIONS:Based on nucleotide and amino acid sequences, a high diversity was found among Salmonella bacteriophages which validate the use of prophage sequence analysis as a highly discriminatory subtyping tool for Salmonella. Thorough understanding of the conservation and variation of prophage genomic characteristics will facilitate their rational design and use as tools for bacterial strain construction, vector development and as anti-bacterial agents.