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

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

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

Managing Contamination and Diverse Bacterial Loads in 16S rRNA Deep Sequencing of Clinical Samples: Implications of the Law of Small Numbers

In this article, we investigate patterns of microbial DNA contamination in targeted 16S rRNA amplicon sequencing (16S deep sequencing) and demonstrate how this can be used to filter background bacterial DNA in diagnostic microbiology. We also investigate the importance of sequencing depth. We first determined the patterns of contamination by performing repeat 16S deep sequencing of negative and positive extraction controls. This process identified a few bacterial species dominating across all replicates but also a high intersample variability among low abundance contaminant species in replicates split before PCR amplification. Replicates split after PCR amplification yielded almost identical sequencing results. On the basis of these observations, we suggest using the abundance of the most dominant contaminant species to define a threshold in each clinical sample from where identifications with lower abundances possibly represent contamination. We evaluated this approach by sequencing of a diluted, staggered mock community and of bile samples from 41 patients with acute cholangitis and noninfectious bile duct stenosis. All clinical samples were sequenced twice using different sequencing depths. We were able to demonstrate the following: (i) The high intersample variability between sequencing replicates is caused by events occurring before or during the PCR amplification step. (ii) Knowledge about the most dominant contaminant species can be used to establish sample-specific cutoffs for reliable identifications. (iii) Below the level of the most abundant contaminant, it rapidly becomes very demanding to reliably discriminate between background and true findings. (iv) Adequate sequencing depth can be claimed only when the analysis also picks up background contamination

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

The Finnish new variant of Chlamydia trachomatis escaping detection in the Aptima Combo 2 assay is widespread across Norway, June to August 2019

The ‘Finnish new variant of Chlamydia trachomatis’ (FI-nvCT), escaping detection in the Aptima Combo 2 assay (AC2), is widespread across Norway. From June to August 2019, 84% (81/97) of available AC2/Aptima CT discordant samples from five laboratories were confirmed as FI-nvCT. Two additional CT variants (CT 23S rRNA C1514T and G1523A) also escaped AC2 detection. The high FI-nvCT proportion might indicate a long-term national spread and it cannot be excluded that FI-nvCT emerged in Norway