Background/Objectives: Biofilm formation by Acinetobacter baumannii contributes to its persistence in clinical settings and resistance to antibiotic treatment. This study aims to identify and characterize antimicrobials from lactic acid bacteria (LAB) using molecular and in silico approaches that can prevent and disrupt A. baumannii biofilms, assess their antimicrobial host range, and define their synergy with current antibiotics. Methods: Thirteen LAB isolates from the Human Microbiome Project were screened for anti-biofilm activity against A. baumannii. Conditioned media was further tested against six ESKAPE pathogens and three skin commensals. Lentilactobacillus hilgardii was selected for detailed study and antimicrobial peptide (AMP) prediction analysis due to limited toxicity toward commensals. In silico identified peptides were synthesized and tested individually and in combination with sub-MIC doses of an antibiotic. Results: Conditioned media from five LAB species (Lentilactobacillus hilgardii, Lentilactobacillus buchneri, Ligilactobacillus ruminis, Limosilactobacillus fermentum, and Limosilactobacillus antri) significantly inhibited A. baumannii biofilm formation and reduced biomass of mature biofilms. LAB-conditioned media also exhibited broad-spectrum activity against ESKAPE pathogens, though effects on commensals varied. Bioinformatically predicted AMPs from L. hilgardii inhibited planktonic A. baumannii growth but showed no direct biofilm activity even at high doses. However, AMPs were found to synergize with sub-MIC doses of meropenem against mature biofilms leading to decolonization. Conclusions: Our study provides a comprehensive platform for the discovery and characterization of AMPs and supports using commensal bacteria to reduce, prevent, and decolonize biofilms from pathogenic bacteria in community and nosocomial settings.

This study investigates the efficacy of conditioned media derived from lactic acid bacteria (LAB) in preventing and disrupting Acinetobacter baumannii biofilms. Of the 13 LAB isolates from the Human Microbiome Project tested, five distinct species Lentilactobacillus hilgardii, Lentilactobacillus buchneri, Ligilactobacillus ruminis, Limosilactobacillus fermentum, and Limosilactobacillus antri demonstrated significant prevention of A. baumannii biofilm formation and reduced the biomass of pre-formed mature biofilms. The study extended to the six ESKAPE pathogens and three skin commensals, demonstrating broad-spectrum antimicrobial activity of LAB conditioned media against ESKAPE pathogens, albeit with variable effects on skin commensals. L. hilgardii was characterized further due to its reduced toxicity against skin commensals. Fractionation studies indicated potential peptide involvement; thus, bioinformatic analysis was used to predict antimicrobial peptides (AMPs) within the L. hilgardii genome, which were synthesized and tested, showing effective inhibition of A. baumannii planktonic growth. However, these peptides did not affect biofilms at concentrations up to 30 µg/mL, although synergy with sub-MIC levels of meropenem significantly disrupted mature biofilms. Our study provides a comprehensive platform for the discovery and characterization of AMPs and support the notion of how commensals can be used to reduce, prevent and decolonize biofilms from pathogenic bacteria in community and nosocomial settings.

Serratia marcescens is an opportunistic pathogen that causes severe hospital-acquired infections, notable for its biofilm formation abilities and development of extensive antibiotic resistance. Here we evaluated the efficacy of bacteriophages, antibiotics, and antimicrobial peptides (BAP), alone and in combination, against fourteen multi-drug-resistant (MDR) S. marcescens isolates sourced from hospitals and other environmental settings in an in vitro biofilm model. Phage combination with a cocktail of sub-minimal inhibitory concentration (MIC) of penicillin-streptomycin, kanamycin, and ciprofloxacin, reduced biofilm biomass, however, complete decolonization was not achieved. Incorporating an antimicrobial peptide cocktail into this regimen eradicated 99.99% of multi-drug-resistant isolates grown planktonically or in surface-associated biofilms. Microscopy and viability assays confirmed extensive biofilm disruption and bacterial clearance without regrowth. These findings reveal that simultaneous interference of cell wall synthesis, protein translation, DNA replication, and membrane integrity can overcome S. marcescens antimicrobial defenses, establishing a multifaceted therapeutic framework for managing device-associated infections caused by MDR pathogens.

Background/Objectives: Serratia marcescens is an opportunistic pathogen that causes severe hospital-acquired infections, notable for its biofilm formation abilities and development of extensive antibiotic resistance. Here, we aim to evaluate the efficacy of bacteriophages, antibiotics, and antimicrobial peptides (BAP), alone and in combination, against fourteen multidrug-resistant (MDR) S. marcescens isolates sourced from hospitals and other environmental settings. Methods: S. marcescens was grown planktonically or in surface-associated biofilms, and biofilm biomass was measured via changes in absorbance and colony-forming units or live/death staining. Results: Combining bacteriophage with a low-dose cocktail of penicillin–streptomycin, kanamycin, and ciprofloxacin enhanced antimicrobial activity compared with antibiotics alone. Across the isolate panel, responses to BAP treatment varied according to determined antibiotic resistance profiles. The highly resistant AR-0517 isolate was selected for detailed mature biofilm analysis, where the BAP treatment reduced biofilm biomass by 97.8% and recoverable bacteria by 99.99%. Microscopy and viability assays further confirmed extensive biofilm disruption and bacterial killing. Conclusions: These findings demonstrate that simultaneous targeting of multiple bacterial pathways can enhance antimicrobial activity against MDR S. marcescens in vitro and support further evaluation of BAP as a potential strategy for biofilm-associated infections.