Kalimuthusamy Natarajaseenivasan

Leptospirosis, a major issue in veterinary medicine and public health, is a bacterial systemic disease caused by pathogenic spirochetes of the genus Leptospira. This infectious disease affects a wide range of animal species, including humans, indicating its widespread impact. Leptospires are highly diverse, with over 20 species, which are divided into over 300 serovars and more than 24 serogroups, which complicates the pathogenicity of the disease. Leptospirosis causes economic losses in livestock, which has larger consequences for food security and farming communities. Leptospirosis develops differently in livestock and wildlife, with small mammals, especially rodents, playing a crucial role as maintenance hosts. Effective management requires surveillance systems that employ serological, molecular, and cultural approaches. The interdependence of human and animal health emphasizes the need for a worldwide One Health approach that prioritizes collaborative work, research, and targeted interventions to address the multifaceted challenges of leptospirosis in a unified manner.

Candida species, normally part of the healthy human flora, can cause severe opportunistic infections when their population increases. This risk is even greater in immunocompromised individuals. Women using intrauterine contraceptive devices (IUDs) are at higher risk for IUD-associated vulvovaginal candidiasis (VVC) because the device provides a surface for biofilm formation. This biofilm formation allows the normal flora to become opportunistic pathogens, leading to symptoms of VVC such as hemorrhage, pelvic pain, inflammation, itching and discharge. VVC is often linked to IUD use, requiring the prompt removal of these devices for effective treatment. This study evaluated the activity of the arginine-rich peptide “protamine” against Candida albicans, Candida tropicalis and Candida krusei isolated from IUD users who had signs of VVC. The antimicrobial activity was measured using the agar disk diffusion and microbroth dilution methods to determine the minimum inhibitory concentration (MIC). The MIC values of protamine against C. albicans, C. tropicalis and C. krusei are 32 μg mL⁻¹, 64 μg mL⁻¹ and 256 μg mL⁻¹, respectively. The determined MIC of protamine was used for a biofilm inhibition assay by crystal violet staining. Protamine inhibited the biofilm formation of the VVC isolates, and its mechanisms were studied through scanning electron microscopy (SEM) and a reactive oxygen species (ROS) assay. The disruption of cell membranes and the induction of oxidative stress appear to be key mechanisms underlying its anti-candidal effects. The results from an in vitro assay support the potential use of protamine as an antibiofilm agent to coat IUDs in the future for protective purposes.

Background/Objective: Indwelling intrauterine contraceptive devices (IUDs) have surfaces that facilitate the attachment of Candida spp., creating a suitable environment for biofilm formation. Due to this, vulvovaginal candidiasis (VVC) is frequently linked to IUD usage, necessitating the prompt removal of these devices for effective treatment. In this study, we evaluated the susceptibility of antimicrobial peptides in vitro against biofilm forming, Amphotericin B (MIC50 > 2 mg L⁻¹) resistant Candida krusei and Candida tropicalis isolated from IUD users who had signs of vaginal candidiasis (hemorrhage, pelvic pain, inflammation, itching, and vaginal discharge). Three antimicrobial peptides, namely, epinecidin-1 (epi-1) and its two variants, namely, variant-1 (Var-1) and variant-2 (Var-2), which were reported to have enhanced antibacterial activity were tested against IUD isolates (C. krusei and C. tropicalis) with pathogenic form of Candida albicans as control. Variants of epi-1, namely, Var-1 and Var-2 were created by substituting lysine in the place of histidine and alanine. Methods: The antimicrobial activity was measured using the microbroth dilution method to determine the minimum inhibitory concentration (MIC) of peptides against C. albicans, C. krusei and C. tropicalis. The MIC of each peptide was used for biofilm assay by Crystal violet staining, Scanning Electron Microscopy, and Reactive Oxygen Species (ROS) assay. To find the possible mechanism of anti-biofilm activity by the peptides, their ability to interact with Candida spp. cell membrane proteins such as Exo-β-(1,3)-Glucanase, Secreted Aspartic Proteinase (Sap) 1, and N-terminal Domain Adhesin: Als 9-2 were determined through PatchDock. Results: The MIC values of peptides: epi-1, var-1 and var-2 against C. albicans are 128 μg mL⁻¹, 64 μg mL⁻¹ and 32 μg mL⁻¹, C. tropicalis are 256 μg mL⁻¹, 64 μg mL^−1, and 32 μg mL⁻¹ and C. krusei are 128 µg mL⁻¹, 128 µg mL⁻¹ and 64 µg mL⁻¹, respectively. Both the variants outperformed epi-1. Specifically for tested Candida spp., var-1 showed two- to four-fold enhancements and var-2 showed two- to eight-fold enhancements compared to epi-1. Electron microscopy confirmed that the mechanism of action involves pore formation thus inducing reactive oxygen species in Candida spp. cell membrane. Computational analysis showed that the peptides have a high tendency to interact with Candida spp. cell membrane proteins such as Exo-β-(1,3)-Glucanase, Secreted Aspartic Proteinase (Sap) 1, and N-terminal Domain Adhesin: Als 9-2, thereby preventing biofilm formation. Conclusions: The in vitro evidence supports the potential use of epi-1 and its variants to be used as an anti-biofilm agent to coat IUDs in the future for therapeutic purposes.

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