The aim of this study was to determine the epidemiology (location, microbial load, microbiome, presence/absence of biofilm and pathogens, including ESKAPE—Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species, and antimicrobial susceptibility profiles) of the bacterial contamination on intensive care units (ICUs) surfaces. Fifty‐seven high‐touched surfaces were collected from adult, paediatric and neonatal ICUs from two large public Brazilian hospitals from central and north regions. Samples (c. 4 cm²) were subjected to culture (qualitative), qPCR targeting 16s rRNA gene (microbial load—bacteria per cm²), 16s rRNA amplicon sequencing (microbiome analysis) and scanning electron (SEM) or confocal laser scanning microscopy (CLSM) (biofilm presence). Multidrug resistant organisms (MROs) were detected using specific chromogenic agar. The average bacterial load was 1·32 × 10⁴ bacteria per cm², container for newborn feeding bottles, stretcher mattress, humidicrib mattress filling and computer keyboards presented the higher bioburden. However, only 45·6% (26/57) were culture‐positive, including 4/26 with MROs. ESKAPE organisms were detected in 51·8% of the samples subjected to next‐generation sequencing. Viability staining and CLSM demonstrated live bacteria on 76·7% of culture‐negative samples. Biofilm was present on all surfaces subjected to microscopy (n = 56), demonstrating that current cleaning practices are suboptimal and reinforcing that MROs are incorporated into hospital surfaces biofilm. Significance and Impact of the Study Contamination of healthcare facilities surfaces has been shown to play a major role in transmission of pathogens. The findings of this study show that dry surface biofilms are widespread and can incorporate pathogens and multidrug‐resistant organisms (MROs). Biofilms on highly touched surfaces pose a risk to patients, as dry surface biofilms persist for long period and micro‐organisms within biofilm have been shown to be transmitted. This study also provides a better understanding of microbial populations in hospital environments, reinforcing that pathogens and MROs are found incorporated into biofilm, which impacts the difficulty in cleaning/disinfection.

Organisms causing healthcare associated infections can be sourced from the inanimate environment around patients. Residing in a biofilm increases the chances of these organisms persisting in the environment. We aimed to characterise bacterial environmental contamination, genetically and physiologically, and relate this to general intensive care unit (ICU) cleanliness. Cleanliness was determined by adenosine triphosphate (ATP) measurements of 95 high-touch objects. Bacteriological samples were obtained from the same sites (n=95) and from aseptically removed sections (destructive samples, n=20). Bacterial enrichment culture was conducted using tryptone soya broth prior to plating on horse blood agar, MacConkey agar, and screening chromogenic agar for identification of multidrug resistance organism (MDRO). Bacterial load and microbial diversity were determined using quantitative PCR (qPCR) and next generation DNA sequencing respectively. Confocal laser scanning microscopy and scanning electron microscopy were used to visually confirm the biofilm presence. Many intensive care surfaces (61%) were highly contaminated by biological soil as determined by ATP bioluminescence testing. The degree of biological soiling was not associated with bacterial contamination as detected by qPCR. Bacterial load ranged from 78.21 to 3.71×108 (median=900) bacteria/100cm2. Surface swabs from 71/95 sites (75%) were culture-positive; of these 16 (22.5%) contained MDRO. The most abundant genera were Staphylococcus, Propionibacterium, Pseudomonas, Bacillus, Enterococcus, Streptococcus and Acinetobacter. Biofilm was visually confirmed by microscopy on 70% (14/20) of items. Bacterial biofilms and MDROs were found on ICU surfaces despite regular cleaning in Saudi Arabia, suggesting that biofilm development is not controlled by current cleaning practices.