dMORECOVERY: A Swab-Based Surface Sampling Protocol Incorporating a Nutrient-Free Resuscitation Step for the Detection of Clinically Relevant Gram-Negative Pathogens in the Viable but Non-Culturable State

Difficult-to-treat Gram-negative bacteria are a major cause of healthcare-associated infections due to multidrug resistance and limited therapeutic options. The hospital environment plays a central role in the persistence and transmission of infection, making environmental monitoring an essential component of infection prevention and control plans. Standard surface sampling techniques, including swabs, contact plates, and sponges, are widely used for environmental surveillance and are all based on culture-dependent methods. However, these techniques may underestimate the actual level of bacterial contamination since they fail to detect bacteria in the viable but non-culturable (VBNC) state, a reversible physiological condition in which bacterial cells remain viable but do not grow on conventional culture media. An innovative environmental sampling protocol, herein named MORECOVERY, has been developed to improve the detection of VBNC bacteria. The protocol integrates an essential resuscitation step, which improves the recovery of VBNC Gram-negative bacteria by a few orders of magnitude, into the standard swab-based sampling workflow. Following sample collection, swabs are incubated for 24 h at 37 °C in a carbon-free resuscitation buffer before plating, enabling the recovery of VBNC bacterial pathogens that would otherwise remain undetectable. By improving the recovery of VBNC cells, the MORECOVERY protocol allows a more accurate assessment of bacterial contamination of critical surfaces in healthcare settings. Its simplicity and minimal variation from standard workflows facilitate easy implementation in routine environmental monitoring. Key features • Enables the detection of Gram-negative bacteria in the viable but non-culturable (VBNC) state, allowing the recovery of otherwise undetectable bacterial contaminants. • Is compatible with standard swab-based environmental sampling workflows and routine microbiological procedures. • Relies on low-cost materials and standard laboratory equipment and can easily be implemented in healthcare facilities and other settings requiring careful monitoring of bacterial contamination. • Enhances the sensitivity of culture-based environmental surveillance methods through a simple resuscitation step prior to plating. • Combines a carbon-free resuscitation buffer and 24-h incubation at 37 °C, preventing bacterial growth while ensuring VBNC resuscitation without bacterial replication.