Metagenomic and Phenotypic Insights Into Biofilm‐Forming Pathogens in Patients With Nosocomial Sepsis

Biofilm-related infections significantly contribute to bacterial diseases, with estimates suggesting that at least 80% of such infections are associated with biofilms. These infections often involve opportunistic pathogens, which not only influence the type of infection but also impact the microenvironment by interacting with other polymicrobial pathogens, thereby altering microbial diversity within the infection site. The present study was designed to assess potential changes in bacterial communities across various infection types. The 50 samples were collected and pooled from different anatomical locations: II-H1 (calf), ul-H2 (thighs), ft-H3 (upper leg), ct-H4 (chest), and Ca-H5 (catheter). The 16S rDNA sequencing was performed on 10 representative samples using the Sanger method to identify bacterial taxa, whereas the metagenomic analysis was conducted on the Illumina MiSeq platform (Illumina, Inc., San Diego, California). Sanger sequencing identifying several bacterial strains including Bacterium MS-AsIII-61, Bacterium HB33-1, Mammaliicoccus sciuri SSB38, multiple Staphylococcus species (S. aureus DA101 and S8, Staphylococcus sp. C0021-01R and TSA25S, S. cohnii FC2265, and S. saprophyticus A), and Enterobacter hormaechei D15. The metagenomics analysis revealed variations and diversity in the different location across the organ by relative abundance of 5 bacterial phyla and 38 species. The Proteobacteria phylum was the most abundant phylum across all sites, with the highest prevalence observed in Ca-H5, followed by ul-H2, ct-H4, II-H1, and ft-H3 in the decreasing order. In contrast, the Bacteroidetes phylum exhibited the highest abundance in ft-H3. Catheter-associated infections (Ca-H5 site) show a homogeneous ARG profile, dominated by genes supporting biofilm formation and persistence. MSA samples reflect diversity in methicillin and multidrug resistance genes, consistent with surgical-site and opportunistic infections. Trypto samples may represent an environmental or experimental condition leading to alternative ARG expression, highlighting site- or condition-specific variations. The different virulence factor responsible for the boost in the establishment of biofilms in these pathogens includes, surface adhesion proteins, increasing resilience to environmental, efflux pumps, quorum-sensing regulators, stresses, and antibiotic treatments. The study demonstrates the dynamic nature and impact of biofilm-related infections at anatomical sites. It also focused on biofilm-associated infections at surgical sites, their progression into chronic conditions, and the corresponding treatment patterns. The integration of metagenomic analysis with phenotypic studies provided deeper insights into the roles of key genes and their mechanisms in biofilm formation.