# ESKAPE Gram-negative bacteria escape culture-based detection upon desiccation on abiotic surfaces

## TL;DR

This study shows that dangerous hospital bacteria can survive on surfaces in a hidden state, escaping detection by standard methods, and suggests a new approach to improve detection.

## Contribution

The study reveals that Gram-negative ESKAPE pathogens can enter a viable but non-culturable state on hospital surfaces and can be resuscitated for detection.

## Key findings

- All ESKAPE pathogens showed reduced cultivability after desiccation on abiotic surfaces.
- Gram-negative species could be resuscitated, while Gram-positive species could not.
- Resuscitation improved detection of VBNC Gram-negative bacteria compared to standard methods.

## Abstract

ESKAPE bacteria, namely Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., are leading causes of hospital-acquired infections and a major therapeutic challenge due to multidrug resistance. Hospital surfaces and medical devices are critical reservoirs for the transmission of these pathogens to patients. Standard methods for detecting microorganisms in the hospital environment are culture-based, so they cannot identify bacteria in the viable but non-culturable (VBNC) state. VBNC bacteria remain metabolically active and potentially infectious, but they fail to grow in conventional, nutrient-rich culture media. Reversion from the VBNC to the cultivable state is termed resuscitation. To assess whether ESKAPE species enter the VBNC state upon desiccation on abiotic materials commonly utilized in clinical facilities and can be resuscitated, bacterial cells were desiccated for 1 week on glass, different plastics, cotton, and titanium surfaces, then resuscitated in a carbon-free buffer. After desiccation, all ESKAPE pathogens exhibited reduced cultivability, with species- and surface-dependent variability. Gram-positive ESKAPE species did not regain cultivability after resuscitation. Conversely, Gram-negative species reverted to the cultivable state, indicating a transition to the VBNC state in response to desiccation. Compared to the standard methodology for biocontamination control (EN 17141:2020), the resuscitation step prior to culture yielded a significantly greater recovery of Gram-negative ESKAPE bacteria in the VBNC state from both experimentally contaminated samples and environmental surfaces. These findings pose the need for environmental monitoring approaches capable of detecting VBNC pathogens on abiotic hospital surfaces.

Accurate detection of microbial contamination in the hospital environment is fundamental for preventing nosocomial infections. Current protocols for environmental surveillance, however, rely almost exclusively on culture-based methods, which overlook bacteria in the viable but non-culturable (VBNC) state. This study demonstrates that clinically relevant Gram-negative ESKAPE pathogens can persist on hospital surfaces in the VBNC state, thereby evading conventional approaches for environmental control, resulting in substantial underestimation of the bacterial burden. We further show that a simple resuscitation step restores the cultivability of VBNC cells, improving their recovery rate, ultimately resulting in much greater sensitivity compared with conventional biocontamination control methods. These findings reveal a critical limitation of current environmental surveillance approaches and highlight the importance of integrating VBNC detection into monitoring protocols for achieving a more accurate assessment of surface contamination to strengthen infection prevention strategies.