title: Sequencing of bacterial microflora in peripheral blood: our experience with HIV-infected patients.
abstract: The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development of the humoral and cellular mucosal immune system. Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities. HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and bacterial DNA fragments, which contribute to systemic immune activation. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy. We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene followed by a sequencing analysis. Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 16S ribosomial gene. Following PCR product purification, cloning and sequencing analyses are performed.
authors.affiliation: Department of Medicine, Surgery and Dentistry, Clinic of Infectious Diseases, San Paolo Hospital University of Milan, Italy.
authors.forename: Esther
authors.initials: E
authors.lastname: Merlini
authors.forename: Giusi M
authors.initials: GM
authors.lastname: Bellistri
authors.forename: Camilla
authors.initials: C
authors.lastname: Tincati
authors.forename: Antonella
authors.initials: A
authors.lastname: d'Arminio Monforte
authors.forename: Giulia
authors.initials: G
authors.lastname: Marchetti
mesh_terms: D016022:Case-Control Studies; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D041981:Gastrointestinal Tract / Q000276:immunology / Q000382:microbiology* / Q000821:virology; D015658:HIV Infections / Q000097:blood* / Q000276:immunology / Q000382:microbiology*; D006801:Humans; D018928:Immunity, Mucosal / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S