title: Analysis and description of the stages of Aspergillus fumigatus biofilm formation using scanning electron microscopy.
abstract: Biofilms are a highly structured consortia of microorganisms that adhere to a substrate and are encased within an extracellular matrix (ECM) that is produced by the organisms themselves. Aspergillus fumigatus is a biotechnological fungus that has a medical and phytopathogenic significance, and its biofilm occurs in both natural and artificial environments; therefore, studies on the stages observed in biofilm formation are of great significance due to the limited knowledge that exists on this specific topic and because there are multiple applications that are being carried out.
  
  
  
  Growth curves were obtained from the soil and clinical isolates of the A. fumigatus biofilm formation. The optimal conditions for both of the isolates were inocula of 1 × 106conidia/mL, incubated at 28 °C during 24 h; these showed stages similar to those described in classic microbial growth: the lag, exponential, and stationary phases. However, the biofilms formed at 37 °C were uneven. The A. fumigatus biofilm was similar regardless of the isolation source, but differences were presented according to the incubation temperature. The biofilm stages included the following: 1) adhesion to the plate surface (4 h), cell co-aggregation and exopolymeric substance (EPS) production; 2) conidial germination into hyphae (8-12 h), development, hyphal elongation, and expansion with channel formation (16-20 h); and 3) biofilm maturation as follows: mycelia development, hyphal layering networks, and channels formation, and high structural arrangement of the mycelia that included hyphal anastomosis and an extensive production of ECM (24 h); the ECM covered, surrounded and strengthened the mycelial arrangements, particular at 37 °C. In the clinical isolate, irregular fungal structures, such as microhyphae that are short and slender hyphae, occurred; 4) In cell dispersion, the soil isolate exhibited higher conidia than the clinical isolate, which had the capacity to germinate and generate new mycelia growth (24 h). In addition, we present images on the biofilm's structural arrangement and chemical composition using fluorochromes to detect metabolic activity (FUNI) and mark molecules, such as chitin, DNA, mannose, glucose and proteins.
  
  
  
  To our knowledge, this is the first time that, in vitro, scanning electronic microscopy (SEM) images of the stages of A. fumigatus biofilm formation have been presented with a particular emphasis on the high hyphal organization and in diverse ECM to observe biofilm maturation.
authors.affiliation: Laboratorio de Micología Médica, Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Carpio y Plan de Ayala s/n, Col. Casco de Santo Tomás, Del. Miguel Hidalgo, 11340, Mexico City, Mexico.
authors.forename: Alejandra Itzel
authors.initials: AI
authors.lastname: González-Ramírez
authors.affiliation: Unidad de Microscopía ENCB, Instituto Politécnico Nacional (IPN), 11340, Mexico City, Mexico.
authors.forename: Adrián
authors.initials: A
authors.lastname: Ramírez-Granillo
authors.affiliation: Laboratorio de Micología Médica, Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Carpio y Plan de Ayala s/n, Col. Casco de Santo Tomás, Del. Miguel Hidalgo, 11340, Mexico City, Mexico.
authors.forename: María Gabriela
authors.initials: MG
authors.lastname: Medina-Canales
authors.affiliation: Laboratorio de Micología Médica, Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Carpio y Plan de Ayala s/n, Col. Casco de Santo Tomás, Del. Miguel Hidalgo, 11340, Mexico City, Mexico. avrodriguez@hotmail.com.
authors.forename: Aída Verónica
authors.initials: AV
authors.lastname: Rodríguez-Tovar
authors.affiliation: Laboratorio de Micología Médica, Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Carpio y Plan de Ayala s/n, Col. Casco de Santo Tomás, Del. Miguel Hidalgo, 11340, Mexico City, Mexico. angeles.12mar@hotmail.com.|Prolongación de Carpio y Plan de Ayala s/n, C.P. 11340, Ciudad de México, Mexico. angeles.12mar@hotmail.com.
authors.forename: María Angeles
authors.initials: MA
authors.lastname: Martínez-Rivera
mesh_terms: D001228:Aspergillosis / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000166:cytology* / Q000254:growth & development / Q000302:isolation & purification / Q000502:physiology*; D018441:Biofilms / Q000254:growth & development*; D003470:Culture Media; D005109:Extracellular Matrix / Q000382:microbiology / Q000502:physiology; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics; D018525:Germination / Q000502:physiology; D006801:Humans; D025301:Hyphae / Q000166:cytology / Q000254:growth & development; D008800:Mexico; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D012988:Soil Microbiology; D013172:Spores, Fungal / Q000166:cytology / Q000254:growth & development; D013696:Temperature
chemical_list: D003470:Culture Media; D005656:Fungal Proteins
keywords: Aspergillus fumigatus biofilm; Microhyphae; Scanning electronic microscopy (SEM); Stages of biofilm