Peer Review Report For: Biofilm formation in carbapenem-resistant Acinetobacter baumannii: Investigating the prevalence of the pgaABCD locus in clinical isolates [version 1; peer review: 1 approved, 1 approved with reservations]

Background Carbapenem-resistant Acinetobacter baumannii (CRAB) is an important nosocomial pathogen with high biofilm-forming ability and multidrug resistance. This study aimed to investigate the prevalence of the pgaABCD locus in clinical CRAB isolates and its relationship with biofilm production and antimicrobial resistance. Methods A total of 150 non-duplicate clinical A. baumannii isolates were collected from healthcare facilities in Shahrekord, Iran. Identification was performed by PCR and biochemical tests. Antimicrobial susceptibility was determined using disk diffusion, and biofilm formation was assessed by the crystal violet method. PCR was used to detect pgaABCD genes. Hemolytic and proteolytic activities were evaluated on blood and skim milk agar. Statistical analyses included chi-square tests and heatmap clustering of resistance profiles. Results Results showed that all isolates were multidrug-resistant, showing the highest resistance to ciprofloxacin (100%), ceftazidime (90%), and cefepime (86.66%). Of these, 90 (60%) were CRAB, all forming biofilms, with 55.5% producing strong biofilms. Hemolytic activity occurred in 14% of isolates and was significantly associated with strong biofilm formation (p < 0.0001). Proteolytic activity was observed in all isolates without significant association. The pgaA and pgaD genes were detected in 100% of isolates, while pgaB and pgaC were found in 84.4% and 93.3%, respectively. Strong biofilm formation correlated significantly with pgaB (p < 0.0001) and pgaC (p = 0.0002). Heatmap analysis showed higher antibiotic resistance in strong biofilm producer s . Conclusion Strong biofilm formation in CRAB is associated with pgaB / pgaC genes and hemolysin production, highlighting the importance of anti-biofilm strategies and molecular monitoring in clinical settings.