Nanopore Sequencing Allows Recovery of High-Quality Completely Closed Genomes of All Cronobacter Species from Powdered Infant Formula Overnight Enrichments

Precision metagenomic approaches using Oxford Nanopore Technology (ONT) sequencing has been shown to allow recovery of complete genomes of foodborne bacteria from overnight enrichments of agricultural waters. This study tests the applicability of a similar approach for <i>Cronobacter</i> genome recovery from powdered infant formula (PIF) overnight enrichments, where <i>Cronobacter</i> typically dominates the overall microbiome (>90%). This study aimed to test whether using ONT sequencing of overnight PIF enrichments could recover a completely closed <i>Cronobacter</i> genome for further genomic characterization. Ten PIF samples, each inoculated with different <i>Cronobacter</i> strains, covering <i>Cronobacter sakazakii</i>, <i>C. muytjensii</i>, <i>C. dublinensis</i>, <i>C. turicensis</i>, and <i>C. universalis</i>, were processed according to the Bacteriological Analytical Manual (BAM) protocol. Real-time quantitative PCR (qPCR) was used for initial screening (detection and quantification) of the overnight enrichments and confirmed that the inoculated PIF samples after the overnight enrichment had high levels of <i>Cronobacter</i> (10⁷ to 10⁹ CFU/mL). DNA from overnight PIF enrichments was extracted from the enrichment broth and sequenced using ONT. Results showed that ONT sequencing could accurately identify, characterize, and close the genomes of <i>Cronobacter</i> strains from overnight PIF enrichments in 3 days, much faster than the nearly 2 weeks required by the current BAM method. Complete genome recovery and species differentiation were achieved. This suggests that combining qPCR with ONT sequencing provides a rapid, cost-effective alternative for detecting and characterizing <i>Cronobacter</i> in PIF, enabling timely corrective actions during outbreaks.