Filamentous surface structures drive biofilm formation in ICU-isolated <i>Acinetobacter baumannii</i> , <i>Pseudomonas aeruginosa</i> , and <i>Staphylococcus aureus</i> : implications for persistent environmental contamination

The relationship between surface biofilms and the persistent contamination of environmental surfaces has garnered increasing attention. Although biofilm research is extensive, systematic comparisons of the morphological characteristics of pathogens isolated from healthcare surfaces, particularly strains that retain biofilm-forming capacity after long-term preservation, are still lacking. In this study, we aimed to investigate the biofilm formation potential and ultrastructural features of the cell membrane surfaces of healthcare environment-isolated <i>Acinetobacter baumannii</i>, <i>Pseudomonas aeruginosa</i>, and <i>Staphylococcus aureus</i>. The revived strains were inoculated into 96-well plates containing TSB and incubated for 24 h to assess their biofilm-forming potential under favorable growth conditions. Biofilm formation was assessed using crystal violet staining. Strains with different biofilm-forming abilities were examined for their morphological characteristics under a scanning electron microscope. Of the 198 historically preserved strains of the three common pathogens, 162 were successfully revived (78 <i>A. baumannii</i>, 36 <i>P. aeruginosa</i>, and 48 <i>S. aureus</i>). The biofilm formation rates of <i>A. baumannii</i>, <i>P. aeruginosa</i>, and <i>S. aureus</i> were 70.51%, 88.89%, and 25%, respectively. The cell surface morphology between the biofilm- and non-biofilm-forming strains differed significantly. Biofilm-forming strains exhibited numerous filamentous structures on their surfaces and displayed aggregation and multidimensional stacking owing to the "net-like" effect of the filaments. In contrast, non-biofilm-forming strains had smooth surfaces without filamentous structures or aggregation. This study provides systematic evidence for the biofilm-forming capabilities of common pathogens associated with healthcare-associated infections, isolated from healthcare environment surfaces. All biofilm-forming strains displayed distinct filamentous structures on their surfaces, with the clonal strains exhibiting similar characteristics.IMPORTANCEThis study is the first to demonstrate the biofilm-forming capacity and morphological characteristics of <i>Acinetobacter baumannii</i>, <i>Pseudomonas aeruginosa</i>, and <i>Staphylococcus aureus</i> in intensive care unit (ICU) environments, filling a critical gap in our understanding of biofilm mechanisms among healthcare-associated pathogens. Notably, <i>P. aeruginosa</i> exhibited an 88.89% biofilm formation rate, with its distinctive filamentous fibrous structures significantly enhancing bacterial adhesion and aggregation-a key explanation for persistent environmental contamination. These findings directly inform the optimization of ICU cleaning protocols, promote the development of biofilm-targeted disinfection standards, and provide a scientific foundation for refining environmental monitoring metrics in infection control policies, ultimately reducing healthcare-associated infection rates.