Characterization of abscesses from liver, pancreas and kidney using deep sequencing of the 16S rRNA gene

To characterize the microbial communities in abscess material from liver, pancreas, and kidneys, we performed deep sequencing of the 16S rRNA gene, in addition to cultivation and Sanger based 16S rRNA gene sequencing directly from the samples. Fifty-nine abscess samples were investigated, 38 from liver, 11 from pancreas, 10 from kidney. Using deep sequencing we made 227 bacterial identifications in 52 specimens, as compared to 69 identifications from the 44 specimens positive by culture. Escherichia coli, Enterococcus sp., Klebsiella sp. and Streptococcus sp. were the most common findings, but various anaerobe bacteria also constituted a large part of the microflora and those were frequently not detected by culture. Culture-independent methods like 16S deep sequencing can significantly improve microbiological diagnostics of clinical specimens. They are particularly valuable for complex purulent infections like abdominal abscesses. Therefore, deep sequencing approaches should be considered as a part of the available repertoire in diagnostic hospital laboratories.publishedVersio

Bacteria and fungi in acute cholecystitis. A prospective study comparing next generation sequencing to culture

Objectives: Guidelines for antibiotic treatment of acute cholecystitis are based on studies using culture techniques for microbial identification. Microbial culture has well described limitations and more comprehensive data on the microbial spectrum may support adjustments of these recommendations. We used next generation sequencing to conduct a thorough microbiological characterization of bile-samples from patients with moderate and severe acute cholecystitis. Methods: We prospectively included patients with moderate and severe acute cholecystitis, undergoing percutaneous or perioperative drainage of the gall bladder. Bile samples were analyzed using both culture and deep sequencing of bacterial 16S rRNA and rpoB genes and the fungal ITS2-segment. Clinical details were evaluated by medical record review. Results: Thirty-six patients with moderate and severe acute cholecystitis were included. Bile from 31 (86%) of these contained bacteria (29) and/or fungi (5) as determined by sequencing. Culture identified only 40 (38%) of the 106 microbes identified by sequencing. In none of the 15 polymicrobial samples did culture detect all present microbes. Frequently identified bacteria often missed by culture included oral streptococci, anaerobic bacteria, enterococci and Enterobacteriaceae other than Klebsiella spp. and Escherichia coli. Conclusions: Culture techniques display decreased sensitivity for the microbial diagnostics of acute cholecystitis leaving possible pathogens undetected.publishedVersio

Investigating the human jejunal microbiota

Descriptions of the small intestinal microbiota are deficient and conflicting. We aimed to get a reliable description of the jejunal bacterial microbiota by investigating samples from two separate jejunal segments collected from the luminal mucosa during surgery. Sixty patients with morbid obesity selected for elective gastric bypass surgery were included in this survey. Samples collected by rubbing a swab against the mucosa of proximal and mid jejunal segments were characterized both quantitatively and qualitatively using a combination of microbial culture, a universal quantitative PCR and 16S deep sequencing. Within the inherent limitations of partial 16S sequencing, bacteria were assigned to the species level. By microbial culture, 53 patients (88.3%) had an estimated bacterial density of < 1600 cfu/ml in both segments whereof 31 (51.7%) were culture negative in both segments corresponding to a bacterial density below 160 cfu/ml. By quantitative PCR, 46 patients (76.7%) had less than 104 bacterial genomes/ml in both segments. The most abundant and frequently identified species by 16S deep sequencing were associated with the oral cavity, most often from the Streptococcus mitis group, the Streptococcus sanguinis group, Granulicatella adiacens/para-adiacens, the Schaalia odontolytica complex and Gemella haemolysans/taiwanensis. In general, few bacterial species were identified per sample and there was a low consistency both between the two investigated segments in each patient and between patients. The jejunal mucosa of fasting obese patients contains relatively few microorganisms and a core microbiota could not be established. The identified microbes are likely representatives of a transient microbiota and there is a high degree of overlap between the most frequently identified species in the jejunum and the recently described ileum core microbiota.publishedVersio

Pulmonary phthalate exposure and asthma - is PPAR a plausible mechanistic link?

Due to their extensive use as plasticisers in numerous consumer products, phthalates have become ubiquitous environmental contaminants. An increasing number of epidemiological studies suggest that exposure to phthalates may be associated with worsening or development of airway diseases. Peroxisome Proliferation Activated Receptors (PPAR)s, identified as important targets for phthalates in early studies in rodent liver, have been suggested as a possible mechanistic link. In this review we discuss the likelihood of an involvement of PPARs in asthma development and exacerbation due to pulmonary phthalate exposure. First, we go through the literature on indoor air levels of phthalates and pulmonary phthalate kinetics. These data are then used to estimate the pulmonary phthalate levels due to inhalation exposure. Secondly, the literature on phthalate-induced activation or modulation of PPARs is summarized. Based on these data, we discuss whether pulmonary phthalate exposure is likely to cause PPAR activation, and if this is a plausible mechanism for adverse effects of phthalates in the lung. It is concluded that the pulmonary concentrations of some phthalates may be sufficient to cause a direct activation of PPARs. Since PPARs mainly mediate anti-inflammatory effects in the lungs, a direct activation is not a likely molecular mechanism for adverse effects of phthalates. However, possible modulatory effects of phthalates on PPARs deserve further investigation, including partial antagonist effects and/or cross talk with other signalling pathways. Moreover other mechanisms, including interactions between phthalates and other receptors, could also contribute to possible adverse pulmonary effects of phthalates

Improved 6-year overall survival in AT/RT - results of the registry study Rhabdoid 2007

Atypical teratoid rhabdoid tumors (AT/RT) are characterized by mutations and subsequent inactivation of SMARCB1 (INI1, hSNF5), a predilection for very young children and an unfavorable outcome. The European Registry for rhabdoid tumors (EU-RHAB) was established to generate a common European database and to establish a standardized treatment regimen as the basis for phase I/II trials. Thus, genetic analyses, neuropathologic and radiologic diagnoses, and a consensus treatment regimen were prospectively evaluated. From 2005 to 2009, 31 patients with AT/RT from four countries were recruited into the registry study Rhabdoid 2007 and treated with systemic and intraventricular chemotherapy. Eight patients received high-dose chemotherapy, 23 radiotherapy, and 17 maintenance therapy. Reference evaluations were performed in 64% (genetic analyses, FISH, MLPA, sequencing) up to 97% (neuropathology, INI1 stain). Germ-line mutations (GLM) were detected in 6/21 patients. Prolonged overall survival was associated with age above 3years, radiotherapy and achievement of a complete remission. 6-year overall and event-free survival rates were 46% (+/- 0.10) and 45% (+/- 0.09), respectively. Serious adverse events and one treatment-related death due to insufficiency of a ventriculo peritoneal shunt (VP-shunt) and consecutive herniation were noted. Acquisition of standardized data including reference diagnosis and a standard treatment schedule improved data quality along with a survival benefit. Treatment was feasible with significant but manageable toxicity. Although our analysis is biased due to heterogeneous adherence to therapy, EU-RHAB provides the best available basis for phase I/II clinical trials

Bruk av massiv parallell sekvensering for påvisning og identifikasjon av mikrober i galle hos pasienter med akutt kolangitt

De fleste infeksjonssykdommer som forårsakes av bakterier og sopp kan behandles med antimikrobielle midler. Kjennskap til hvilke mikrober som forårsaker sykdommene er viktig for å kunne gi pasienten riktig behandling. Den vanligste metoden for påvisning og karakterisering av bakterier og sopp er dyrkning. Dyrkningsmetoden har imidlertid vesentlige begrensninger, da den er avhengig av levende mikrober som er i stand til å vokse under de betingelsene som kan tilbys i laboratoriet. Vi har derfor etablert en dyrkningsuavhengig metode for identifisering av bakterier og sopp, basert på massiv parallell sekvensering av utvalgte gener. Sekvensering av 16S rRNA-genet er en godt etablert metode for identifisering av bakterier i diagnostiske laboratorier, og kan i mange tilfeller identifisere bakterier til artsnivå. Det finnes imidlertid unntak, blant annet for arter innen Enterobacteriaceae-familien og innen Enterococcus-, Streptococcus- og Staphylococcus-slektene. For å løse dette har vi derfor innført sekvensering av rpoB-genet, som et supplement til 16S rRNA. Sekvensering av ITS-2 segmentet er inkludert for å kunne påvise og identifisere sopp. Tjue galleprøver fra nitten pasienter med kolecystitt og kolangitt ble undersøkt, og det ble totalt funnet 35 ulike arter. Det ble påvist signifikant flere mikrober ved massiv parallell sekvensering enn ved dyrkning, og det var en klar sammenheng mellom mengde påvist DNA og om mikroben lot seg dyrke. Sekvensering av rpoB og ITS-2 ga verdifull tilleggsinformasjon sammenholdt med sekvensering av 16S rRNA alene, og de aller fleste mikrobene lot seg identifisere til artsnivå. Metoden som er etablert i denne studien kan også benyttes til andre typer prøvemateriale, og vil være spesielt verdifull for karakterisering av mikrobiologien ved ulike polymikrobielle infeksjoner

Characterization of abscesses from liver, pancreas and kidney using deep sequencing of the 16S rRNA gene

To characterize the microbial communities in abscess material from liver, pancreas, and kidneys, we performed deep sequencing of the 16S rRNA gene, in addition to cultivation and Sanger based 16S rRNA gene sequencing directly from the samples. Fifty-nine abscess samples were investigated, 38 from liver, 11 from pancreas, 10 from kidney. Using deep sequencing we made 227 bacterial identifications in 52 specimens, as compared to 69 identifications from the 44 specimens positive by culture. Escherichia coli, Enterococcus sp., Klebsiella sp. and Streptococcus sp. were the most common findings, but various anaerobe bacteria also constituted a large part of the microflora and those were frequently not detected by culture. Culture-independent methods like 16S deep sequencing can significantly improve microbiological diagnostics of clinical specimens. They are particularly valuable for complex purulent infections like abdominal abscesses. Therefore, deep sequencing approaches should be considered as a part of the available repertoire in diagnostic hospital laboratories