A fully automated model to form “dry surface biofilms” under optimal dehydration conditions. application to Enterobacteriaceae in healthcare settings

Abstract

Over ten years ago, bacteria attached to surfaces and surrounded by extracellular polymeric substances were observed on dry surfaces in intensive care units. These structures were named "dry surface biofilms" (DSBs). Most in vitro models used to study “DSBs” alternate long hydration phases with short periods of desiccation, producing "semi-dehydrated DSBs" that differ from the conditions in healthcare settings.

Our aim was to create a model that could produce "DSBs" under optimal dehydration conditions and apply it to Enterobacteriaceae. These bacteria are commonly responsible for healthcare-associated infections in our hospital, yet they have received little attention in the context of "DSBs." We developed a fully automated model that mimics the splashing of respiratory secretions by repeatedly nebulizing an inoculum of contaminated artificial saliva. Hydration phases lasted 2 s every 6 h. We investigated the microscopic aspect, mean surface coverage, bacterial culturability and membrane integrity.

After validating the model with methicillin-resistant Staphylococcus aureus (MRSA), we tested wild-type Enterobacter cloacae, wild-type Klebsiella pneumoniae and extensively drug-resistant (XDR) Klebsiella pneumoniae. The latter formed compact dried inocula with the highest surface coverage (29.7 %), containing curled-up bacteria alongside a low number of culturable cells (3 log₁₀). Conversely, dried S. aureus inocula covered a lower surface (10.9 %) but contained more culturable cells (6 log₁₀), which persisted for more than two months. After several weeks of storage, even the samples containing no more culturable bacteria showed bacteria with intact membranes. Subsequent studies must further assess in depth the composition of these deposits and the viability of the bacteria they contain.