Unveiling the In Vitro Antibiofilm Efficacy of Antifungal Lipopeptides Purified from Bacillus sp. against Mixed-Species Biofilms of Candida - Abstract

Sep 29, 2026

Polymicrobial fungal infections are often associated with significant invasive mycosis, with non-albicans Candida species such as Candida glabrata and Candida tropicalis common after C. albicans in biofilm-associated disease. The study evaluates in vitro antibiofilm efficacy of Bacillus-derived lipopeptides AF4 and AF5 against dual-species Candida mixed biofilms across adherence, developmental, and mature phases, showing marked reductions in biofilm biomass and metabolic activity. Microscopy and apoptosis assays (Annexin V-FITC/PI and TUNEL) indicate lipopeptide-driven disruption of biofilms and planktonic forms, including concentration-dependent apoptotic induction and DNA fragmentation, supporting strong antifungal potential.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages17

Reading Time43 min

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Abstract

Introduction

Biofilm formation and polymicrobial interactions

Candida species and biofilm-associated challenges

Study rationale and prior findings

This article is licensed under CC-BY 4.0 [http://pubs.acs.org/journal/acsodf](http://pubs.acs.org/journal/acsodf) Article Unveiling the In Vitro Antibiofilm Efficacy of Antifungal Lipopeptides Purified from Bacillus sp. against Mixed-Species Biofilms of Candida Madhuri Madduri, Shivaprakash M. Rudramurthy, and Utpal Roy* Cite This: ACS Omega 2025, 10, 58671−58687 Read Online | | | | | | | | --- | --- | --- | --- | --- | --- | | ACCESS | Metrics & More | | Article Recommendations | | *sı Supporting Information | ABSTRACT: Polymicrobial fungal infections are often associated with significant invasive mycosis. Candida non-albicans species such as Candida glabrata and Candida tropicalis are most frequent organisms after Candida albicans especially in biofilm-associated fungal infections with significant mortality and morbidity. In the present investigation, the antibiofilm efficacy of Bacillus-derived lipopeptides AF4 and AF5 was assessed against Candida dual species mixed biofilms at different stages such as adherence, developmental, and mature phase in vitro experiments. Interestingly, in this study, C. glabrata was found to be predominant over C. tropicalis, whereas AF4 and AF5 exposures significantly reduced both biofilm biomass and metabolic activity as was revealed through various experiments such a quantification assays and morphological analyses. The advanced microscopy suggests the strong biofilm-busting activity of both lipopeptides. Additionally, the elucidation of their mode of action against the planktonic phase of C. glabrata and C. tropicalis was performed to investigate the presence of stages of apoptosis with Annexin V-FITC/propidium iodide staining and TUNEL assays. Two strains were exposed to sub-MIC and minimum inhibitory concentration (MIC), also with 2× MIC of AF4 and AF5. A significant shift from live cells to early stage and late-stage apoptotic induction percentage was increased along with increasing the concentration of 1× MIC to 2× MIC. TUNEL results confirmed the DNA fragmentation by the enhanced green fluorescence intensity in cells in TUNEL-positive nuclei. Taken together, our results demonstrate that Bacillus-derived lipopeptides AF4 and AF5 hold great promise with strong antibiofilm properties. The overall antibiofilm properties and mode of action characterization studies unequivocally confirm the antifungal potential of the two investigational lipopeptides, with the ability to challenge several fungal infections. 1. INTRODUCTION Candida glabrata and Candida tropicalis are listed as high prioritized fungal pathogens according to World Health Organization 2022.1 Candidiasis, a fungal infection, causes both superficial and systemic mycosis with high morbidity and mortality.2 Candida species are human commensal fungal organisms, very well-known for causing opportunistic fungal infections in immunity compromised hosts especially hospitalized patients.3 Non-albicans Candida species such as C. tropicalis, C. glabrata, and Candida parapsilosis most commonly coisolated fungal pathogens after Candida albicans. According to the fungal nomenclature, C. glabrata is currently known as Nakaseomyces glabratus or Nakaseomyces glabrata and previously known as Torulopsis glabrata.4,5 C. glabrata can potentially disseminate throughout the host system6 and can cause infection with a significant mortality rate. Besides, C. glabrata is endowed with the uncanny ability to adhere and to develop biofilms on host tissues which render the infections clinically challenging since the cells possessed by biofilms happen to be intrinsically resistant to several antifungal treatments.7 The environmental adaptability of these species increases the adherence to the biotic and abiotic surfaces, and forming colonization is one of the major virulent factors associated with biofilm infections in humans.8−10 Candida biofilms possess a significant challenge in the realm of human health, specifically due

FAQs

Which Candida species and biofilm types were investigated in the study?

The work examined mixed-species Candida biofilms, focusing on Candida glabrata and Candida tropicalis, including dual-species interactions across different biofilm stages.

How were the Bacillus-derived lipopeptides AF4 and AF5 evaluated for antibiofilm effects?

AF4 and AF5 were tested in vitro against the biofilm phases (adherence, developmental, and mature), using assays to quantify biofilm biomass and metabolic activity, along with morphological and microscopy analyses.

What evidence supports apoptosis and DNA damage in response to AF4 and AF5?

The study used Annexin V-FITC/propidium iodide staining to assess early and late apoptotic induction and TUNEL assays to confirm DNA fragmentation via enhanced fluorescence in TUNEL-positive nuclei.

Sep 29, 2026

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