Purpose: The purpose of this study was to identify the mechanism of autoaggregation in C. sakazakii ATCC 29544 (CS29544).
Methods: CS29544 was grown aerobically with agitation at 37°C. Stationary phase cells were stagnantly incubated at 25°C for 2 h to observe autoaggregation. Stable non-autoaggregative CS29544 were isolated following successive passages (6 total) of the uppermost bacterial cells. The resulting non-autoaggregators and CS29544 were sequenced using the Illumina platform to identify single nucleotide polymorphisms (SNPs). Candidate genes (containing SNPs) were identified by BLAST and confirmed by Sanger sequencing. Candidate genes were disrupted in the CS29544 wild-type using the lambda Red recombinase system. Presence or absence of flagella was determined by either flagella stain or transmission electron microscopy.
Results: Initially, CS29544 was found to autoaggregate within 30 min; however, the growth media didn’t fully clarify. Using selective enrichment, we isolated two independent non-autoaggregating variants of CS29544. Comparison of the variant genomes to the wild-type genome revealed unique SNPs in two flagella proteins; flhA and fliG. The disruption of flhA and fliG, along with fliC, in the wild-type resulted in non-autoaggregating CS29544 that did not possess extracellular flagella. Disruption of the motor genes, motAB, didn’t affect autoaggregation. In conclusion, autoaggregation in CS29544 is mediated by the presence of structurally-intact flagella.
Significance: Understanding the mechanism of autoaggregation in C. sakazakii will provide novel insights into how C. sakazakii initiates formation of biofilms and colonization of the gastrointestinal tract.