Lactoferrin, chitosan and Melaleuca alternifolia-natural products that show promise in candidiasis treatment

Abstract The evolution of microorganisms resistant to many medicines has become a major challenge for the scientific community around the world. Motivated by the gravity of such a situation, the World Health Organization released a report in 2014 with the aim of providing updated information on this critical scenario. Among the most worrying microorganisms, species from the genus Candida have exhibited a high rate of resistance to antifungal drugs. Therefore, the objective of this review is to show that the use of natural products (extracts or isolated biomolecules), along with conventional antifungal therapy, can be a very promising strategy to overcome microbial multiresistance. Some promising alternatives are essential oils of Melaleuca alternifolia (mainly composed of terpinen-4-ol, a type of monoterpene), lactoferrin (a peptide isolated from milk) and chitosan (a copolymer from chitin). Such products have great potential to increase antifungal therapy efficacy, mitigate side effects and provide a wide range of action in antifungal therapy.

Lactoferrina bovina disminuye la invasión de Salmonella enterica en células HEp-2 / Bovine lactoferrin decreases the invasion of Salmonella enterica to HEp-2 cells

Objetivo: Evaluar el efecto de la lactoferrina bovina (Lfb) en el proceso de invasión de Salmonella enterica ser. Typhimurium en células HEp-2. Materiales y métodos: Se infectaron células HEp-2 con 10(6) unidades formadoras de colonia (UFC) de la bacteria en ausencia y presencia de 1 y 10 mg/mL de Lfb (saturada con hierro) durante 1,5 horas a 37°C. En este estudio evaluamos 2 tratamientos: pre-infección (las células HEp-2 se incubaron con Lf 1 hora, previo a la infección con Salmonella) y post-infección (la Lfb se adicionó 15 minutos después de la infección). La capacidad de invasión de Salmonella se determinó mediante la cuantificación de las UFC recuperadas desde el interior de las células HEp-2 (después del tratamiento con 100 μg/mL y 10 μg/mL de gentamicina y Triton X-100). Resultados: En el tratamiento pre-infección se observó una disminución de 23% en la invasión de Salmonella cuando las células HEp-2 fueron pre-incubadas con 1 mg/mL de Lfb (2,8x10(5) vs 2,1x10(5), p=0,04) y una disminución de 50% cuando fueron pre-incubadas con 10 mg/mL de Lfb (2,8x10(5) vs 1,4x10(5), p=0,04). Con el tratamiento post-infección no se observaron cambios en la capacidad de invasión de Salmonella. Conclusiones: Los resultados indican que Lfb reduce la capacidad de invadir de Salmonella enterica ser. Typhimurium a células HEp-2 en el tratamiento pre-infección

Objective: To assess the effect of bovine lactoferrin (bLf) on the invasion of Salmonella enterica ser. Typhimurium to HEp-2 cells. Materials and methods: HEp-2 monolayers were infected with 10(6) colony forming unit (CFU) of bacteria in the absence and presence of 1 and 10 mg/mL of bLf (iron-saturated) and incubated 1.5 hours at 37°C. Two treatments were evaluated: preinfection (HEp-2 cells were incubated with bLf one hour prior to infection with Salmonella) and post-infection (bLf was added 15 minutes after the infection). Invasiveness of Salmonella was determined throgh quantification of CFU recovered from inside the HEp-2 cells (after treatment with 100 μg/mL and 10 μg/mL of gentamicin and Triton X -100). Results: In the pre-infection treatment, we observed a decrease of 23% of Salmonella invasion when HEp-2 cells were pre incubated with 1 mg/mL of bLf (2.8x105 vs 2.1x105, p=0.04) and 50% when them were pre-incubated with 10 mg/mL of bLf (2.8x10(5) vs 1.4x10(5), p=0.04). In post-infection treatment, no changes were observed in the invasiveness of Salmonella. Conclusion: The results indicated that bLf reduces the invasiveness of Salmonella enterica ser. Typhimurium to HEp-2 cells in the pre-infection treatment

Antimicrobial Activity and Synergism of Lactoferrin and Lysozyme Against Cariogenic Microorganisms

The present study evaluated the antimicrobial in vitro effects of the salivary proteins lactoferrin and lysozyme on microorganisms involved in the carious process, obtaining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Streptococcus mutans (ATCC 25175) and Lactobacillus casei (ATCC 7469) were submitted to broth macrodilution of lysozyme at 80 mg/mL and lactoferrin at 200 mg/mL. The tubes were read in a spectrophotometer after they had been incubated at 37 °C for 18 h, in a carbon dioxide chamber, in order to read the MIC. A new subculture was carried on agar plates to obtain the MBC. The agar diffusion method was also tested, using BHI agar with 100 µL of the standardized microbial inocula. Filter-paper disks soaked in 10 µL of the solutions lactoferrin (200 µg/mL) and lysozyme (80 µg/mL) were placed on the agar surface. Inhibition halos were not observed on the plates, showing the absence of the antimicrobial effects of these proteins in this method. The bactericidal and bacteriostatic effects of lysozyme on L. casei were 50.3 mg/mL and 43.1 mg/mL respectively. The bactericidal and bacteriostatic effects on S. mutans were 68.5 mg/mL and 58.7 mg/mL. Lactoferrin did not induce any inhibitory effects on any microorganism, even in the concentration of 200 mg/mL. There was not a synergic antimicrobial effect of proteins, when they were tested together, even in the concentration of 42.8 mg/mL of lysozyme and 114 mg/mL of lactoferrin (the highest values evaluated). S. mutans and L. casei were only inhibited by lysozyme, not affected by lactoferrin and by the synergic use of both proteins.

O presente estudo avaliou, in vitro, o efeito antimicrobiano das proteínas salivares lactoferrina e lisozima sobre micro-organismos envolvidos no processo carioso, obtendo suas concentrações inibitórias mínimas (CIM) e bactericidas mínimas (CBM). Cepas de Streptococcus mutans (ATCC 25175) e Lactobacillus casei (ATCC 7469) foram submetidas a macrodiluição em caldo das soluções de lisozima a 80 mg/mL e lactoferrina a 200 mg/mL. A leitura dos tubos foi realizada em espectrofotômetro, após a incubação a 37 °C por 18 h em estufa de CO2, para verificação da CIM. Uma nova subcultura foi semeada em placas de ágar para a obtenção da CBM. O método de difusão em ágar foi também testado utilizando-se placas de Petri com ágar BHI com 100 µL do inóculo microbiano padronizado. Discos de filtro de papel embebidos com 10 µL das soluções de lactoferrina (200 µg/mL) e lisozima (80 µg/mL) foram colocados sobre a superfície do ágar. Não foi observado halo de inibição nas placas, demonstrando ausência de efeito antimicrobiano das proteínas neste teste. Os efeitos bactericida e bacteriostático da lisozima sobre L. casei foram 50,3 mg/mL e 43,1 mg/mL respectivamente. Os efeitos bactericida e bacteriostático sobre S. mutans foram 68,5 mg/mL e 58,7 mg/mL. A lactoferrina não induziu nenhum efeito inibitório sobre nenhuma bactéria, mesmo na concentração de 200 mg/mL. Não houve efeito antimicrobiano sinérgico das proteínas, quando testadas conjuntamente, e mesmo até em concentrações de 42,8 mg/mL de lisozima e 114 mg/mL de lactoferrina (os maiores valores avaliados). S. mutans e L. casei foram inibidos somente pela lisozima, não sendo afetados pela lactoferrina e pelo uso sinérgico de ambas proteínas.

Biofilm biomass disruption by natural substances with potential for endodontic use

This study evaluated the in vitro effects of four natural substances on the biomass of bacterial biofilms to assess their potential use as root canal irrigants. The following substances and their combinations were tested: 0.2% farnesol; 5% xylitol; 20% xylitol; 0.2% farnesol and 5% xylitol; 0.2% farnesol, 5% xylitol, and 0.1% lactoferrin; 5% xylitol and 0.1% lactoferrin; and 20 mM salicylic acid. The crystal violet assay was used to evaluate the effects of these substances on the biomass of biofilms formed by Enterococcus faecalis and Staphylococcus epidermidis. All substances except for 20 mM salicylic acid and 20% xylitol reduced biofilm mass when compared to controls. The combination of farnesol and xylitol was the most effective agent against E. faecalis ATCC 29212 (p < 0.05). Farnesol combined with xylitol and lactoferrin was the most effective against biofilms of the endodontic strain of E. faecalis MB35 (p < 0.05). Similarly, combinations involving farnesol, xylitol, and lactoferrin reduced the biomass of S. epidermidis biofilms. In general, farnesol, xylitol, and lactoferrin or farnesol and xylitol reduced biofilm biomass most effectively. Therefore, it was concluded that combinations of antibiofilm substances have potential use in endodontic treatment to combat biofilms.

Antimicrobial action of lactoferrin on Staphylococcus aureus inoculated in Minas frescal cheese

The effect of the addition of lactoferrin on Staphylococcus aureus to Minas frescal cheese was evaluated. Three cheeses were made: control (S. aureus), Lf-2% (2% lactoferrin + S. aureus) and Lf-4% (4% lactoferrin + S. aureus). Cheeses were packaged and S. aureus populations were determined on days 1, 8, 15, 22 and 29 of storage at 6°C. The experiment was repeated three times. S. aureus population increased 1.3 logarithmic cycles in the control cheese during storage, while it decreased to numbers below the detection limit in cheeses containing lactoferrin, over the same period. Moreover, antimicrobial effect showed to be dose-dependent.

Ação antimicrobiana de lactoferrina sobre Staphylococcus aureus inoculado em queijo Minas frescal. O efeito da adição de lactoferrina sobre a viabilidade de Staphylococcus aureus inoculado em queijo Minas frescal foi avaliado. Três queijos foram produzidos: controle (S. aureus), Lf-2% (2% lactoferrina + S. aureus) e Lf-4% (4% lactoferrina + S. aureus). Os queijos foram armazenados e as populações de S. aureus foram determinadas durante o armazenamento refrigerado a 6°C nos dias 1, 8, 15, 22 e 29. Os experimentos foram realizados em triplicata. As populações de S. aureus aumentaram 1,3 ciclos logarítmicos nos queijos controle durante o armazenamento, enquanto que essas populações foram reduzidas abaixo do limite de detecção nos queijos contendo lactoferrina no mesmo período. Além disso, observou-se que o efeito antimicrobiano foi dose-dependente.