Article Figures & Data
Figures
- FIG 1
Bacteria found in AOM samples and strengths of their signals. The vertical axis shows individual samples: /2, second sample from the subject; PERF, sample from perforation; TS, sample from tympanostomy tube; other samples are from myringotomy; c, culture; neg., culture negative; Sp, S. pneumoniae; Hi, H. influenzae; Mc, M. catarrhalis; Sa, Staphylococcus aureus; other, other bacteria. The horizontal axis shows the nine most abundant species or genera that exceeded 3% of the total sample sequencing signal in at least two AOM samples. The intersections are the relative abundances of the total sequencing signal as percentages (rounded to the nearest integer). Taxa comprising <3% of the total sample sequencing signal were disregarded. The assignation of species by 16S rRNA gene profiling is simplified, since the 16S profiling of the V4 region could not distinguish between closely related species in several instances, as follows. (i) S. pneumoniae and the less frequent S. pseudopneumoniae; the latter is commonly (mis)identified as S. pneumoniae by clinical microbiology laboratories worldwide. The sequence of the profiled V4 region of the 16S rRNA gene is also closely related to those of several other streptococci. (ii) H. influenzae and the less frequent H. haemolyticus; the latter could be excluded in culture-positive cases by its beta hemolysis. (iii) M. catarrhalis and the less frequent Moraxella nonliquefaciens; the two could be differentiated only by classic microbiological techniques, including differences in typical antibiograms. Finally, (iv) the 16S profiles in the V4 region are identical in many Staphylococci; please see the text for methods that disentangled the signals.
- FIG 2
Quantities of the bacteria assessed using the proportions within the individual sample profiles. Negs, count of samples that were negative for the bacterium in the 16S profiling, i.e., had a quantity lower than 3% of the profile signal.
- FIG 3
Comparison of detection by specific real-time PCR and by 16S profiling. Horizontal axis, threshold cycle of the respective specific PCR; vertical axis, proportion of the overall profiling signal within the sample. Note that the V4 sequence of Streptococcus pneumoniae is nearly identical to those of several further streptococci (e.g., Streptococcus dentisani, Streptococcus tigurinus, Streptococcus oralis, Streptococcus mitis, and Streptococcus infantis). These are most likely present in several samples, denoted by crosses along the vertical axis of the top left panel: here, the pneumococcus-specific real-time PCR test using the autolysin gene (lytA) is negative, but the weak signal in 16S profiling indicates the presence of these streptococci.
- FIG 4
Workflow of the study. *, data from specific PCR tests of these pathogens come from our previous study (12).
Tables
- TABLE 1
Bacteria found in the 16S profiles
Finding in the bacterial profilea No. of samples positive for the species (n = 90) No. of positive samples % of all samples Streptococcus pneumoniae 28 31 As a dominant pathogenb 14 16 Sole finding but <50% of signalc 3 3.3 Nondominant part of mixed florad 11 12 Haemophilus influenza 24 27 As a dominant pathogen 15 17 Sole finding but <50% of signal 3 3.3 Nondominant part of mixed flora 6 6.7 Moraxella catarrhalis 18 20 As a dominant pathogen 5 5.6 Sole finding but <50% of signal 4 4.4 Nondominant part of mixed flora 9 10 Staphylococcus spp. 21 23 As a dominant pathogen 3 3.3 Sole finding but <50% of signal 6 6.7 Nondominant part of mixed flora 12 13 Turicella otitidis 5 5.6 As a dominant pathogen 2 2.2 Sole finding but <50% of signal 1 1.1 Nondominant part of mixed flora 2 2.2 Alloiococcus otitidis 3 3.3 As a dominant pathogen 0 0 Sole finding but <50% of signal 0 0 Nondominant part of mixed flora 3 3.3 Other bacteria not listed above 14 16 As a dominant pathogen 0 0 Sole finding but <50% of signal 3e 3.3 Nondominant part of mixed flora 11f 12 No clear bacterial finding 14 16 No bacterium found 11 12 Undetermined species, <5% of signal 3 3.3 ↵a The bacteria originating from the PCR components (Taq polymerase) are not shown.
↵b A dominant pathogen was defined as a bacterium that makes up half or more of the total 16S rRNA gene profile.
↵c Bacterium occupying 3.0 to 49% of the sequencing signal; no other bacteria were detectable over the threshold 3.0% signal except the contaminant signal from Taq polymerase.
↵d Bacterium occupying 3 to 49% of the sequencing signal; also, other bacteria were present in the profile at >3.0%.
↵e Prevotella melaninogenica (4% in sample from patient 41 and 6% in sample from patient 48) and undetermined Sphingobacterium (8% in patient 72). All three samples were taken by myringotomy.
↵f Prevotella melaninogenica (31% of the profile of sample from patient 73, 3% in patient 22, and 4% in patient 50), Veillonella dispar (20% in patient 73 and 10% in patient 72), Veillonella montpellierensis (13% in patient 67, 6% in patient 73, and 4% in patient 2), Lactococcus lactis (7% in patient 15 and 7% in second sample from patient 59), Corynebacterium tuberculostearicum (4% from patient 50 and 3% from patient 70, both in samples with dominant S. pneumoniae), and undetermined Sphingobacterium.
FIG S1
Bacterial DNA found in negative controls. The overall number of 6 no-template controls (water instead of MEF subjected to extraction and all subsequent analytic steps) was expanded into 17 control positions in the second round of amplification. Data presented here are collapsed back into the six original samples. Download FIG S1, PDF file, 0.1 MB.
Copyright © 2017 Sillanpää et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .
TABLE S1
Primers used for verification of selected OTUs found in 16S profiles from the present study. Download TABLE S1, PDF file, 0.05 MB.
Copyright © 2017 Sillanpää et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .
