Purpose: The objective of this study was to investigate molecular mechanisms which may contribute to desiccation tolerance in L. monocytogenes.
Methods: A library of 11,700 Himar1 L. monocytogenes 568 transposon insertion mutants was initially screened in a microplate assay for strains displaying increased or decreased survival when desiccated in TSB at 43% RH and 15°C. The desiccation phenotypes were subsequently assessed on food grade stainless steel coupons. Sequencing of arbitrary PCR products was used to determine transposon insertion sites in strains exhibiting > 0.5 log CFU/cm2 change in survival after seven days of desiccation on the stainless steel coupons. Strain morphology and survival in TSB+20% NaCl was also analyzed.
Results: Initial screening led to the selection of 129 sensitive and 61 tolerant desiccation mutants. Further testing on stainless steel yielded 15 mutants each with increased or decreased desiccation survival compared to the wildtype. Of the 15 sequenced desiccation tolerant mutants, 7 immotile mutants contained Himar1inserts in motility related genes while the remaining mutants harboured inserts in genes involved in fatty acid metabolism, membrane transport, protein biosynthesis, transcription regulation, and virulence. Interrupted genes in the 15 sensitive mutants encode a variety of functions including lipid membrane and protein synthesis, transport, surface and stress proteins. A significant (P = 0.01) positive correlation existed between the survival under desiccation and osmotic stresses, demonstrating cross-protective mechanisms between the two types of water stress.
Significance: This study revealed genes which may contribute to the survival and persistence of L. monocytogenes exposed to low RH conditions in the food processing environment.