title: Development of a magnetic separation method to capture sepsis associated bacteria in blood.
abstract: Bloodstream infections are important public health problems, associated with high mortality due to the inability to detect the pathogen quickly in the early stages of infection. Such inability has led to a growing interest in the development of a rapid, sensitive, and specific assay to detect these pathogens. In an effort to improve diagnostic efficiency, we present here a magnetic separation method for bacteria that is based on mutated lysozyme (LysE35A) to capture S. aureus from whole blood. LysE35A-coated beads were able to bind different MSSA and MRSA isolates in the blood and also other six Gram-positive and two Gram-negative species in whole blood. This system was capable to bind bacteria at low concentrations (10CFU/ml) in spiked blood. Samples captured with the mutated lysozyme showed more responsive amplification of the 16S gene than whole blood at concentrations of 10(3)-10(5)CFU. These data demonstrate detection of S. aureus directly in blood samples, without in vitro cultivation. Our results show that capture with LysE35A-coated beads can be useful to develop a point of care diagnostic system for rapid and sensitive detection of pathogens in clinical settings.
authors.affiliation: Instituto de Biologia Molecular do Paraná, Department of Research and Development, Prof. Algacyr Munhoz Mader Street 3775, 81350-010 Curitiba, PR, Brazil. Electronic address: analuisa_kalb@hotmail.com.
authors.forename: Ana Luisa Kalb
authors.initials: ALK
authors.lastname: Lopes
authors.affiliation: Instituto de Biologia Molecular do Paraná, Department of Research and Development, Prof. Algacyr Munhoz Mader Street 3775, 81350-010 Curitiba, PR, Brazil.
authors.forename: Josiane
authors.initials: J
authors.lastname: Cardoso
authors.affiliation: Instituto de Biologia Molecular do Paraná, Department of Research and Development, Prof. Algacyr Munhoz Mader Street 3775, 81350-010 Curitiba, PR, Brazil.
authors.forename: Fernanda Roberta Correa Cleto
authors.initials: FRCC
authors.lastname: Dos Santos
authors.affiliation: Instituto de Biologia Molecular do Paraná, Department of Research and Development, Prof. Algacyr Munhoz Mader Street 3775, 81350-010 Curitiba, PR, Brazil.
authors.forename: Ana Claudia Graziani
authors.initials: ACG
authors.lastname: Silva
authors.affiliation: Instituto de Biologia Molecular do Paraná, Department of Research and Development, Prof. Algacyr Munhoz Mader Street 3775, 81350-010 Curitiba, PR, Brazil.
authors.forename: Maria Isabel
authors.initials: MI
authors.lastname: Stets
authors.affiliation: Laboratory of Proteomic and Protein Engineering, Carlos Chagas Institute, Fiocruz, Prof. Algacyr Munhoz Mader Street 3775, 81350-010 Curitiba, PR, Brazil.
authors.forename: Nilson Ivo Tonin
authors.initials: NIT
authors.lastname: Zanchin
authors.affiliation: Laboratory of Cell Biology, Carlos Chagas Institute, Fiocruz, Prof. Algacyr Munhoz Mader Street 3775, 81350-010 Curitiba, PR, Brazil.
authors.forename: Maurilio José
authors.initials: MJ
authors.lastname: Soares
authors.affiliation: Instituto de Biologia Molecular do Paraná, Department of Research and Development, Prof. Algacyr Munhoz Mader Street 3775, 81350-010 Curitiba, PR, Brazil.
authors.forename: Marco Aurélio
authors.initials: MA
authors.lastname: Krieger
mesh_terms: D016470:Bacteremia / Q000097:blood / Q000175:diagnosis; D001431:Bacteriological Techniques / Q000379:methods*; D003001:Cloning, Molecular; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000302:isolation & purification*; D008855:Microscopy, Electron, Scanning; D010957:Plasmids / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000302:isolation & purification; D018805:Sepsis / Q000097:blood* / Q000175:diagnosis; D013211:Staphylococcus aureus / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Lysozyme; Magnetic separation; S. aureus; Sepsis