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 β-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 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.

Pathology and Public Health Significance of Salmonella

“…The seriousness of contamination, in the same way as for other irresistible illnesses, relies on insusceptible status, hereditary cosmetics, the specie in question, serovar and status of vaccination against the pathogen (Asmar and Abdel-Haq 2016). Although salmonellosis is seen in most domestic animals, pregnant and lactating young mammals and birds are found to be most susceptible (Wisittipanit et al 2020). Cattle can be chronically infected, and therefore, may serve as carriers within the herd without showing any clinical signs.…”

CRISPR 2 spacer architecture analysis and virulotyping for epidemiological study of Salmonella enterica subsp. Enterica circulating in northern Thailand (2015 -2017)

“…using agarose gel electrophoresis. 28 More recently, CRISPR loci polymorphisms have been utilized in a single-step assay to identify multiple contaminated Salmonella spp. from poultry samples.…”