Worldwide, bacterial resistance is one of the most severe public health problems. Currently, the failure of antibiotics to counteract superbugs highlights the need to search for new molecules with antimicrobial potential to combat them. The objective of this research was to evaluate the antimicrobial activity of Bacillus amyloliquefaciens BS4 against Gram-negative bacteria. Thirty yeasts and thirty-two Bacillus isolates were tested following the agar well-diffusion method. Four Bacillus sp. strains (BS3, BS4, BS17, and BS21) showed antagonistic activity against E. coli ATCC 25922 using bacterial culture (BC) and the cell-free supernatant (CFS), where the BS4 strain stood out, showing inhibitory values of 20.50 ± 0.70 mm and 19.67 ± 0.58 mm for BC and CFS, respectively. The Bacillus sp. BS4 strain can produce antioxidant, non-hemolytic, and antimicrobial metabolites that exhibit activity against several microorganisms such as Salmonella enterica, Klebsiella pneumoniae, Shigella flexneri, Enterobacter aerogenes, Proteus vulgaris, Yersinia enterocolitica, Serratia marcescens, Aeromonas sp., Pseudomonas aeruginosa, Candida albicans, and Candida tropicalis. According to the characterization of the supernatant, the metabolites could be proteinaceous. The production of these metabolites is influenced by carbon and nitrogen sources. The most suitable medium to produce antimicrobial metabolites was TSB broth. The one-factor-at-a-time method was used to standardize parameters such as pH, agitation, temperature, carbon source, nitrogen source, and salts, resulting in the best conditions of pH 7, 150 rpm, 28 °C, starch (2.5 g/L), tryptone (20 g/L), and magnesium sulfate (0.2 g/L), respectively. Moreover, the co-culture was an excellent strategy to improve antimicrobial activity, achieving maximum antimicrobial activity with an inhibition zone of 21.85 ± 1.03 mm. These findings position the Bacillus amyloliquefaciens BS4 strain as a promising candidate for producing bioactive molecules with potential applications in human health.

Bacterial resistance remains a critical global health challenge, driving the continuous search for novel antimicrobial agents. Bacillus amyloliquefaciens is a recognized repository of bioactive metabolites; however, its full biosynthetic potential requires integrated genomic and experimental validation. This study characterized the antimicrobial profile of B. amyloliquefaciens BS4 through a hybrid pipeline. Genome sequencing and de novo assembly revealed a 3.9 Mb chromosome with a G + C content of 46.14%. Functional annotation identified 3,887 coding sequences, including pathways for siderophore biosynthesis and a complete bacilysin biosynthetic cluster. BGC analysis using antiSMASH v7.1.0 and BAGEL4 identified 18 biosynthetic gene clusters, while similarity network analysis via BiG-SCAPE highlighted unique singleton BGCs, indicating untapped biosynthetic diversity. Although in silico screening via Macrel predicted two putative cationic antimicrobial peptides (AMPs), bioactivity-guided purification utilizing sequential RP-HPLC, and de novo sequencing revealed a distinct set of four active peptides. Notably, three of these sequences were identified as fragments derived from the BclA exosporium protein family, highlighting the structural proteome as a non-canonical source of antimicrobials. The purified fractions exhibited activity against M. luteus and E. coli , while displaying no significant hemolytic activity or cytotoxicity, even above the MIC values. Molecular docking further supported the interaction of these candidates with bacterial targets. Overall, this hybrid strategy effectively uncovers the antimicrobial complexity of BS4, revealing ‘cryptic’ peptide candidates with therapeutic potential.

The emergence of antimicrobial resistance (AMR) has driven the search for alternative therapeutic strategies, including antivirulence approaches targeting bacterial quorum sensing (QS). Azelaic acid (AzA), a naturally occurring dicarboxylic acid with known antimicrobial properties, has not previously been characterized as a QS inhibitor in Gram-negative pathogens. This study evaluated the dual antimicrobial and antivirulence activity of AzA against reference strains and clinical isolates of Pseudomonas aeruginosa, Enterobacteriaceae , and Staphylococcus aureus through in vitro assays and molecular docking analyses. Minimum inhibitory concentration (MIC) values ranged from 250 to 1000 µg/mL, with lower MICs observed in clinical isolates of E. coli and S. aureus . Subinhibitory concentrations (250, 500 and 750 µg/mL) were used to assess QS-regulated virulence factors in P. aeruginosa , including pyocyanin, elastase, alginate, and protease production. AzA exhibited a significant, dose-dependent inhibition of all evaluated virulence factors across both reference and multidrug-resistant (MDR) and pan-drug-resistant (PDR) clinical strains (p < 0.001), achieving inhibition levels exceeding 90% in several cases, particularly for protease activity. Molecular docking analyses revealed that AzA interacts with key QS-related proteins (LasI, LasR, PqsD, and PqsR), showing moderate binding affinities (−5.3 to −6.5 kcal/mol) and stable interactions within conserved ligand-binding domains. These findings suggest a multitarget modulatory mechanism affecting interconnected QS pathways. Overall, this study demonstrates, for the first time, that AzA acts as a quorum sensing inhibitor in P. aeruginosa , attenuating virulence without directly affecting bacterial growth, highlighting its potential as a promising antivirulence therapeutic strategy.

Background/Objectives: Antimicrobial resistance (AMR) is a major global health threat that requires the discovery of new antimicrobial agents. Environmental microbiomes from understudied regions represent a valuable source of antimicrobial peptide (AMP) candidates. This study aimed to identify and prioritize AMP candidates from South American genomic and metagenomic datasets and to investigate the antimicrobial potential of bioactive secretomes obtained through bacterial co-culture. Methods: A total of 853 genomes and 360 metagenomes were analyzed using a reproducible genome- and metagenome-mining pipeline combined with machine learning-based AMP prediction. Predicted AMP candidates were further characterized using complementary bioinformatic tools to assess physicochemical, structural, hemolytic, toxicological, anti-inflammatory, and anticancer properties. Selected environmental isolates were subjected to bacterial co-culture, followed by SPE-C18 and HPLC fractionation. Antimicrobial activity, antioxidant activity, hemolysis, minimum inhibitory concentration (MIC), and LC-MS/MS peptidomic analyses were performed on bioactive secretome fractions. Results: Genome and metagenome mining identified diverse AMP candidate sequences associated with bacterial genera including Streptomyces, Bacillus, Burkholderia, and Shewanella. Structural predictions revealed a predominance of α-helical conformations among prioritized candidates. Several secretome fractions obtained from co-cultures displayed antimicrobial activity against Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Active fractions showed no detectable hemolytic activity and exhibited antioxidant activity in DPPH assays. MIC analyses indicated broad-spectrum activity against Escherichia coli ATCC 11229, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii, and MRSA, with an apparent MIC of 10,000 mg/L. LC-MS/MS analysis of bioactive fractions identified peptide sequences by de novo sequencing, including KTESHHK, KRVGPRR, GLFPRLGVSPR, and HHAEHLVHFR. Conclusions: Integrated genome mining, bacterial co-culture activation, and peptidomic analyses provide a useful framework for prioritizing antimicrobial peptide candidates from environmental microbiomes. The identification of peptide-containing bioactive fractions with antimicrobial and antioxidant activities highlights the potential of South American bacterial resources for the discovery of novel antimicrobial compounds. Further purification, peptide synthesis, and biological validation will be required to determine the contribution of individual peptides to the observed activities.