Heat map showing 3 pairs of samples before and after antibiotic treatment with the corresponding clinical characteristics (follow up: 1 day for samples 48/49 and 54/55, 5 days for samples 120/129).

<p>Bacteria were detected in all cases, but in 2 pairs (48–49, 120–129), the composition of bacteria changed, while in one patient (54–55), the composition of bacteria remained the same despite of antibiotic treatment. Blue ellipses indicate culture results. N/A: not available.</p

Bacterial Landscape of Bloodstream Infections in Neutropenic Patients via High Throughput Sequencing

<div><p>Background</p><p>Bloodstream infection (BSI) is a common and potentially life-threatening complication in patients with hematological malignancies and therapy-induced neutropenia. Administration of broad spectrum antibiotics has substantially decreased the mortality rate in febrile neutropenia, but bacterial infection is documented in only one-third or fewer of the cases. BSI is typically diagnosed by blood culture; however, this method can detect only culturable pathogens.</p><p>Methods</p><p>In the present study, a total of 130 blood samples from hematological patients receiving dose-intensive antitumoural treatment were subjected to 16S rRNA PCR and 62 of them were cultured. PCR positive samples were processed to high throughput sequencing by amplifying the V1-V3 regions of the 16S rRNA gene to obtain a full spectrum of bacteria present in BSI.</p><p>Results</p><p>Five phyla and 30 genera were identified with sequencing compared to 2 phyla and 4 genera with culture. The largest proportion of bacteria detected by sequencing belonged to Proteobacteria (55.2%), Firmicutes (33.4%) and Actinobacteria (8.6%), while Fusobacteria (0.4%) and Bacteroidetes (0.1%) were also detected. Ninety-eight percent of the bacteria identified by sequencing were opportunistic human pathogens and 65% belonged to the normal human microbiota.</p><p>Conclusions</p><p>The present study indicates that BSIs in neutropenic hosts contain a much broader diversity of bacteria, likely with host origin, than previously realized. The elevated ratio of Proteobacteria in BSI corroborates the results found in other systemic inflammatory diseases, such as inflammatory bowel disease or mucosal infections. This knowledge may become of value for tailoring antimicrobial drug administration.</p></div

Occurrence of genera in 19 samples identified by sequencing (a). A genus was included if it reached or exceeded 0.5% of the total number of filtered reads in at least one sample. Distribution of OTU-assigned reads per genera in all samples in percentage (b). Diagram shows the pathogenicity and natural habitat of the detected genera based on read percentages (c). Over 96% of the identified reads belonged to opportunistic human pathogens (black stripes), while 64% belonged to the normal human microbiota (grey background).

<p>Occurrence of genera in 19 samples identified by sequencing (a). A genus was included if it reached or exceeded 0.5% of the total number of filtered reads in at least one sample. Distribution of OTU-assigned reads per genera in all samples in percentage (b). Diagram shows the pathogenicity and natural habitat of the detected genera based on read percentages (c). Over 96% of the identified reads belonged to opportunistic human pathogens (black stripes), while 64% belonged to the normal human microbiota (grey background).</p

Representation of the distribution of phyla with sequencing in all samples (a) and in individual samples (b). Detected phyla per sample with blood culture (c).

<p>Representation of the distribution of phyla with sequencing in all samples (a) and in individual samples (b). Detected phyla per sample with blood culture (c).</p

Composition of microbiota from different parts of the body classified by four major bacterial phyla.

<p>The graph was reconstructed based on the data from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135756#pone.0135756.ref044" target="_blank">44</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135756#pone.0135756.ref045" target="_blank">45</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135756#pone.0135756.ref051" target="_blank">51</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135756#pone.0135756.ref053" target="_blank">53</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135756#pone.0135756.ref055" target="_blank">55</a>]. Axes show percentages.</p

Schematic representation of microorganisms detected by sequencing on the species level.

<p>Pink nodes represent the given genera connected to the species. Green lines indicate the most prevalent species. Color lines from Bacteria to genera indicate phyla (Proteobacteria-yellow, Firmicutes-light blue, Actinobacteria-dark blue, Bacteroidetes-purple, Fusobacterium-orange). Species nodes indicate infectious properties: yellow-normal microbiota, red-human pathogen, blue-typically occurs in immunocompromised patients and/or nosocomial infections, white: taxonomy unclear/recently changed or environmental bacteria.</p

Representative amplification curves of 10 experiments show background suppression on a septic blood sample, with blue lines representing the human DNA (β-actin), and pink lines show <i>E. coli</i> DNA amount.

<p>While the amount of human DNA has degraded, the <i>E. coli</i> DNA amount did not change.</p

Study Population Characteristics and Risk Factors for Colonization with ESBL-Producing Bacteria.

1<p>Mid-upper arm circumference at time of enrolment examined on children ≥6 months of age.</p>2<p>Test for linear trend.</p>3<p>Child breastfed at time of enrolment.</p>4<p>Bedsharing with another child <5 years of age.</p>5<p>Number of children <5 years of age living in the same household.</p>6<p>Antibiotic treatment initiated prior to presentation at emergency ward.</p>7<p>Reported antibiotic usage during the month prior to study enrolment (excluding antibiotic usage for current disease).</p>8<p>Child hospitalized ≥1 day during the month prior to enrolment.</p

Carriage Prevalence of ESBL-Producing <i>E. coli</i> and <i>K. pneumoniae</i> According to Age.

<p>Full legend: The ESBL carriage prevalence did not vary depending on age. Absolute numbers are presented within bars.</p

Schematic representation of the presented background suppression method.

<p>In septic blood, the amount of human DNA (black) exceeds pathogen DNA (green) amount (A). The extracted DNA is fragmented, denatured and re-hybridized, then dsDNA is degraded with nucleases (red) specific to double stranded DNA. Since rare DNA re-hybridizes slower, mostly abundant DNA will be degraded.</p