CRISPRSpacerType: Automated CRISPR typing of Cronobacter spp. for contamination tracing and evolutionary analysis via...

Cronobacter spp. are important foodborne pathogens associated with severe neonatal infections and high mortality rates. CRISPR typing's higher resolution than multi-locus sequence typing (MLST) makes it powerful for tracing the source of Cronobacter contamination, yet its broader application is constrained by the technique's complexity. To address this challenge, we developed an automated CRISPR typing tool, CRISPRSpacerType, to perform CRISPR detection, classification, and CRISPR Type (CT) assignment. A local database was constructed for Cronobacter spp. as a reference, and all strains were subsequently typed using this tool. Furthermore, we conducted a detailed analysis of two major pathogenic sequence types, Cronobacter sakazakii ST1 and ST4, to assess the performance of CRISPRSpacerType. A total of 39 and 30 CTs were detected in ST1 and ST4 strains, respectively. Most importantly, significant associations were found linking specific CT types to both predominant colistin antibiotic resistance gene mcr-9.1 carriage and pathogenicity. Spacer analysis revealed potential plasmid transmission and specific phage driven adaptation. Ancestral spacer analysis of ST4 strains further identified a high-risk lineage, which includes both the pathogenic CT2 and mcr-9.1 positive CT325 strains, warranting heightened surveillance. Spacer sequences are closely associated with bacterial evolution and phenotype, making the development of a CRISPR typing tool of high biological significance. CRISPRSpacerType permits the broader application of high resolution CRISPR typing to contamination source tracing and evolutionary monitoring of Cronobacter. This advancement significantly strengthens outbreak prevention and surveillance efforts against this pathogen. Our tool is available from GitHub at https://github.com/zwxyo/CRISPRSpacerType.