Isolation and Functional Characterization of Staphylococcus-Targeting Bacteriophages from Wastewater Around Jakarta

Abstract

Diabetic foot infections (DFIs) are a major complication of diabetes mellitus and are commonly associated with Staphylococcus aureus, a pathogen capable of forming biofilms and exhibiting antibiotic resistance. The increasing prevalence of multidrug-resistant S. aureus highlights the need for alternative antimicrobial strategies. This study aimed to isolate and characterize a lytic bacteriophage targeting S. aureus InaCC B4 from wastewater sources in Jakarta and evaluate its antibacterial and anti-biofilm activities. Bacteriophages were isolated using enrichment culture, spot test screening, and plaque assay titration. Morphological characterization was performed using cryogenic electron microscopy (cryo-EM). Antibacterial activity was evaluated through in vitro killing curve assays at different multiplicities of infection (MOIs), while anti-biofilm activity was assessed using biofilm inhibition and eradication assays quantified by crystal violet staining. Phage stability against commonly used antiseptics was also investigated. A lytic bacteriophage was successfully isolated from Tanah Tinggi wastewater and produced clear plaques with an average diameter of 1.23 ± 0.12 mm. Cryo-EM analysis revealed an icosahedral capsid and a contractile tail, consistent with the morphology of tailed bacteriophages within the class Caudoviricetes. The isolated phage effectively suppressed bacterial growth and demonstrated dose-dependent anti-biofilm activity, achieving up to 57% biofilm inhibition and 80% biofilm eradication at MOI 10. Stability testing showed that the phage remained viable in saline but was completely inactivated after 15 minutes of exposure to 70% ethanol, povidone iodine, and hydrogen peroxide. The results show the potential of the isolated bacteriophage as an alternative antimicrobial and anti-biofilm agent against S. aureus infections.