Genotypic and Phenotypic Characterization of Cronobacter spp. Strains Isolated from Powdered Milk Formulas and Dairy Production Environments

Cronobacter spp. is a pathogenic genus comprising seven species, of which C. sakazakii is particularly notable for its association with neonatal outbreaks linked to powdered infant formula. The severity of infections is associated with virulence factors (VFs) and β-lactam antibiotic resistance genes (ARGs). Next-generation sequencing (NGS) has enabled precise strain typing through core genome multilocus sequence typing (cgMLST), enhancing discrimination and accuracy. This study aimed to use cgMLST (2831 genes) to genomically characterize 34 Cronobacter strains which had been isolated from powdered milk and production surfaces between 2011 and 2022. The identified strains included C. sakazakii ST1, ST4, ST13, ST31 and ST83, as well as C. malonaticus ST60. Overall, there were eight clusters of closely related strains. All strains exhibited resistance to cephalothin, 18 were resistant to ceftazidime and 11 to ampicillin. Various resistance genes (blaCSA, blaCMA, fos, qacJ, marA, AcrAB-TolC, and mcr-9.1) and virulence genes (cpa, nanAKT, fic, relB, fliC) were detected, with some genes being exclusive to C. sakazakii. All strains carried plasmids and mobile genetic elements. The multidrug resistance and presence of virulence genes in these isolates highlight the significant risk that C. sakazakii-contaminated powdered dairy products pose to public health, underscoring the need to adopt proper hygienic manufacturing practices and effectively implement HACCP in their production.