Time-kill curve plot. Peptide LfcinB (20–25)4 against S. aureus ATCC 25923. The peptide was tested at concentrations corresponding to MIC (blue line), 2 MIC (pink line) and 4 MIC (orange line) values.

Palindromic peptide LfcinB (21–25)Pal: RWQWRWQWR was synthesized by Solid Phase Peptide Synthesis (SPPS‐Fmoc/tBu), purified by Reverse Phase Solid Phase Extraction (RP‐SPE) and characterized by Reverse Phase High Performance Liquid Chromatography (RP‐HPLC) and Matrix‐Assisted Laser Desorption/Ionization‐Time Of Flight Mass Spectrometry (MALDI‐TOF MS). The antifungal activity of LfcinB (21–25)Pal against both ATCC strains and clinical isolates of C. albicans, C. glabrata, C. krusei, C. auris and C. tropicalis was evaluated. The palindromic peptide exhibited fungistatic and fungicidal activity against all yeast evaluated. The antifungal activity was dependent on peptide concentration in all cases. Additionally, LfcinB (21–25)Pal (25–50 μg/mL) combined with fluconazole exhibited a synergistic antifungal effect against C. tropicalis 883 and C. krusei 6258 (resistant to fluconazole). This study showed that the palindromic peptide derived from Lactoferricin B (LfcinB) exhibited significant antifungal activity against Candida spp, suggesting that this peptide could have a therapeutic application solely or in combination with fluconazole.

Chimeric peptides containing short sequences derived from bovine Lactoferricin (LfcinB) and Buforin II (BFII) were synthetized using solid‐phase peptide synthesis (SPPS) and characterized via reversed‐phase liquid chromatography and mass spectrometry. The chimeras were obtained with high purity, demonstrating their synthetic viability. The chimeras’ antibacterial activity against Gram‐positive and Gram‐negative strains was evaluated. Our results showed that all the chimeras exhibited greater antibacterial activity against the evaluated strains than the individual sequences, suggesting that chemical binding of short sequences derived from AMPs significantly increased the antibacterial activity. For each strain, the chimera with the best antibacterial activity exerted a bacteriostatic and/or bactericidal effect, which was dependent on the concentration. It was found that: (i) the antibacterial activity of a chimera is mainly influenced by the linked sequences, the palindromic motif RLLRRLLR being the most relevant one; (ii) the inclusion of a spacer between the short sequences did not significantly affect the chimera's synthesis process; however, it enhanced its antibacterial activity against Gram‐negative and Gram‐positive strains; on the other hand, (iii) the replacement of Arg with Lys in the LfcinB or BFII sequences improved the chimeras’ synthesis process without significantly affecting their antibacterial activity. These results illustrate the great importance of the synthesis of chimeric peptides for the generation of promising antibacterial peptides.

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MethodsOver a four-year period, 123 Candida bloodstream isolates were collected at a quaternary care hospital. The isolates were identified by MALDI-TOF MS and their fluconazole (FLC) susceptibility patterns were assessed according to CLSI guidelines. Subsequently, sequencing of ERG11, TAC1 or MRR1, and efflux pump activity were performed for resistant isolates.ResultsOut of 123 clinical strains,C. albicans accounted for 37.4%, followed by C. tropicalis 26.8%, C. parapsilosis 19.5%, C. auris 8.1%, C. glabrata 4.1%, C. krusei 2.4% and C. lusitaniae 1.6%. Resistance to FLC reached 18%; in addition, a high proportion of isolates were cross-resistant to voriconazole. Erg11 amino acid substitutions associated with FLC-resistance (Y132F, K143R, or T220L) were found in 11/19 (58%) of FLCresistant isolates. Furthermore, novel mutations were found in all genes evaluated. Regarding efflux pumps, 8/19 (42%) of FLC-resistant Candida spp strains showed significant efflux activity. Finally, 6/19 (31%) of FLC-resistant isolates neither harbored resistance-associated mutations nor showed efflux pump activity. Among FLC-resistant species, C. auris 7/10 (70%) and C. parapsilosis 6/24 (25%) displayed the highest percentages of resistance (C. albicans 6/46, 13%).DiscussionOverall, 68% of FLC-resistant isolates exhibited a mechanism that could explain their phenotype (e.g. mutations, efflux pump activity, or both). We provide evidence that isolates from patients admitted to a Colombian hospital harbor amino acid substitutions related to resistance to one of the most commonly used molecules in the hospital setting, with Y132F being the most frequently detected.

Due to the increased incidence of fungal infections and the emergence of antifungal resistance mainly by Candida species, the need for safe and effective novel therapies is imperative. Consequently, plants and herbs are a powerful source to combat infections. Here, we evaluated the anti-Candida potential of an ethanolic extract from Piper nigrum. The phytochemical analysis of P. nigrum revealed bioactive compounds such as alkaloids, terpenoids, and tannis. Our results showed that P. nigrum extract suppressed the virulence factors of C. albicans strains, including hyphae formation in both liquid and solid media, reduced secretion of phospholipases/proteinases, and affected biofilm formation. Furthermore, the P. nigrum extract showed no hemolytic effect in vitro and exhibited reduced cytotoxicity on Vero cells and G. mellonella larvae at concentrations that inhibited hyphae and biofilm in C. albicans. Moreover, the extract demonstrated antifungal activity against C. auris strains. In conclusion, the P. nigrum extract affected the growth and morphogenesis of Candida (even in resistant strains), demonstrating that this plant has an anti-candida activity and represents a promising resource for discovering novel antifungal compounds.

Breast cancer is one of the main causes of premature death in women; current treatments have low selectivity, generating strong physical and psychological sequelae. The palindromic peptide R-1-R (RWQWRWQWR) has cytotoxic activity against different cell lines derived from cancer and selectivity against noncancerous cells. To determine if changes in the charge/length of this peptide increase its activity, six peptides were obtained by SPPS, three of them with addition of Arg at the N, C-terminal or both and three with deletion of Arg at the N, C-terminal or both. The cytotoxic and selective activities were evaluated against MCF-7, MDA-MB-231, and MCF-12 cell lines and fibroblast primary cell culture, evidencing that the RR-1-R peptide with the inclusion of Arg in the N-terminal end maintained selectivity and increased cytotoxicity against lines derived from breast cancer. The effect of this addition regarding the type of induced cell death was evaluated by flow cytometry, showing very low rates of necrosis and a significant majority of apoptotic events with activation of both Caspase 8 and Caspase 9. This work allowed us to find a modification that generates a peptide with greater cytotoxic effects and can be considered a promising molecule for other approaches to improve anticancer peptides.

Antimicrobial peptides (AMPs) are considered to be a valuable source for the identification and/or design of promising candidates for the development of antifungal treatments, since they have advantages such as lower tendency to induce resistance, ease of production, and high purity and safety. Bovine lactoferricin (LfcinB) and buforin II (BFII) are AMPs to which great antimicrobial potential has been attributed. The minimum motives with antimicrobial activity derived from LfcinB and BFII are RRWQWR and RLLR, respectively. Nine chimeras containing the minimum motives of both peptides were synthesized and their antifungal activity against fluconazole (FLC)-sensitive and resistant C. albicans, C. glabrata, and C. auris strains was evaluated. The results showed that peptides C9: (RRWQWR)2K-Ahx-RLLRRRLLR and C6: KKWQWK-Ahx-RLLRRLLR exhibited the greatest antifungal activity against two strains of C. albicans, a FLC-sensitive reference strain and a FLC-resistant clinical isolate; no medically significant results were observed with the other chimeras evaluated (MIC ~200 µg/mL). The chimera C6 was also active against sensitive and resistant strains of C. glabrata and C. auris. The combination of branched polyvalent chimeras together with FLC showed a synergistic effect against C. albicans. In addition to exhibiting antifungal activity against reference strains and clinical isolates of Candida spp., they also showed antibacterial activity against both Gram-positive and Gram-negative bacteria, suggesting that these chimeras exhibit a broad antimicrobial spectrum and can be considered to be promising molecules for therapeutic applications.