title: Effect of neem (Azadirachta indica A. Juss) leaf extract on resistant Staphylococcus aureus biofilm formation and Schistosoma mansoni worms.
abstract: BACKGROUND
  There are ethnopharmacological reports supporting the use of neem (Azadirachta indica A. Juss) leaf against bacterial and worm infections. However there is a lack of studies about its effect on bacterial biofilm formation and Schistosoma mansoni worms. This study reports the in vitro effects of neem leaf ethanolic extract (Neem EE) on Methicillin-resistant Staphylococcus aureus (MRSA) biofilm and planktonic aggregation formation, and against S. mansoni worms.
  
  
  METHODS
  Quantification of the Azadirachtin (AZA), thought to be one of their main compounds related to biological effects, was performed. The effect of sub-inhibitory concentrations of Neem EE on biofilm formation and planktonic aggregates of S. aureus was tested using the crystal violet dye method and atomic force microscopy (AFM) analysis, respectively. Changes in S. mansoni motor activity and death of worms were analyzed in vitro after exposition to the extract. Treated schistosomes were also examined using confocal laser scanning microscopy.
  
  
  RESULTS
  It was observed the presence of AZA in the extract (0.14 ± 0.02 mg/L). Testing Neem EE sub-inhibitory concentrations, a significant biofilm adherence inhibition from 62.5 µg/mL for a sensitive S. aureus and 125 µg/mL for two MRSA strains was observed. AFM images revealed that as the Neem EE concentration increases (from 250 to 1000 µg/mL) decreased ability of a chosen MRSA strain to form large aggregates. In relation of anti-schistosoma assay, the extract caused 100% mortality of female worms at a concentration of 50 µg/mL at 72 h of incubation, while 300 µg/mL at 24h of incubation was required to achieve 100% mortality of male worms. The extract also caused significant motor activity reduction in S. mansoni. For instance, at 96 h of incubation with 100 µg/mL, 80% of the worms presented significant motor activity reduction. By the confocal microscopy analysis, the dorsal surface of the tegument of worms exposed to 300 µg/mL (male) and 100 µg/mL (female) of the extract showed severe morphological changes after 24h of treatment.
  
  
  CONCLUSIONS
  Neem leaf ethanolic extract presented inhibitory effect on MRSA biofilm and planktonic aggregation formation, and anthelmintic activity against S. mansoni worms.
authors.affiliation: Biodiversity and Biotechnology Research Center, Biotec, Federal University of Piauí, UFPI, 64202020 Parnaíba, PI, Brazil.
authors.forename: Patrick V
authors.initials: PV
authors.lastname: Quelemes
authors.affiliation: Biodiversity and Biotechnology Research Center, Biotec, Federal University of Piauí, UFPI, 64202020 Parnaíba, PI, Brazil.
authors.forename: Márcia L G
authors.initials: ML
authors.lastname: Perfeito
authors.affiliation: Biodiversity and Biotechnology Research Center, Biotec, Federal University of Piauí, UFPI, 64202020 Parnaíba, PI, Brazil.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Guimarães
authors.affiliation: Biodiversity and Biotechnology Research Center, Biotec, Federal University of Piauí, UFPI, 64202020 Parnaíba, PI, Brazil.
authors.forename: Raimunda C
authors.initials: RC
authors.lastname: dos Santos
authors.affiliation: Biodiversity and Biotechnology Research Center, Biotec, Federal University of Piauí, UFPI, 64202020 Parnaíba, PI, Brazil; Federal University of Vale do São Francisco, UNIVASF, Paulo Afonso, BA, Brazil.
authors.forename: David F
authors.initials: DF
authors.lastname: Lima
authors.affiliation: Laboratory of Parasitology, Instituto Butantan, São Paulo, SP, Brazil.
authors.forename: Carlos
authors.initials: C
authors.lastname: Nascimento
authors.affiliation: Center for Research on Neglected Diseases, University of Guarulhos, Guarulhos, SP, Brazil.
authors.forename: Marcos P N
authors.initials: MP
authors.lastname: Silva
authors.affiliation: Department of Veterinary Morphophysiology, Federal University of Piauí, Teresina, PI 64049550, Brazil.
authors.forename: Maria José dos S
authors.initials: MJ
authors.lastname: Soares
authors.affiliation: Phytobios, Pesquisa, Desenvolvimento e Inovação, Barueri, SP, Brazil.
authors.forename: Cristina D
authors.initials: CD
authors.lastname: Ropke
authors.affiliation: Biodiversity and Biotechnology Research Center, Biotec, Federal University of Piauí, UFPI, 64202020 Parnaíba, PI, Brazil; UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal.
authors.forename: Peter
authors.initials: P
authors.lastname: Eaton
authors.affiliation: Center for Research on Neglected Diseases, University of Guarulhos, Guarulhos, SP, Brazil.
authors.forename: Josué
authors.initials: J
authors.lastname: de Moraes
authors.affiliation: Biodiversity and Biotechnology Research Center, Biotec, Federal University of Piauí, UFPI, 64202020 Parnaíba, PI, Brazil. Electronic address: jrsaleite@gmail.com.
authors.forename: José Roberto S A
authors.initials: JR
authors.lastname: Leite
mesh_terms: D000818:Animals; D000871:Anthelmintics / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D031601:Azadirachta; D001422:Bacterial Adhesion / Q000187:drug effects; D018441:Biofilms / Q000187:drug effects; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects* / Q000254:growth & development / Q000502:physiology; D008826:Microbial Sensitivity Tests; D010936:Plant Extracts / Q000494:pharmacology*; D018515:Plant Leaves; D012550:Schistosoma mansoni / Q000187:drug effects*
chemical_list: D000871:Anthelmintics; D000900:Anti-Bacterial Agents; D010936:Plant Extracts
keywords: Azadirachta indica; Biofilm; Neem leaf extract; Schistosoma mansoni