JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2009, p. 3562–3568
0095-1137/09/$12.00 doi:10.1128/JCM.00973-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 11
Direct 16S rRNA Gene Sequencing from Clinical Specimens, with
Special Focus on Polybacterial Samples and Interpretation of
Mixed DNA Chromatograms䌤
Øyvind Kommedal,1,2* Kristine Kvello,2 Rune Skjåstad,1 Nina Langeland,3,4 and Harald G. Wiker1,2
Department of Microbiology and Immunology, Haukeland University Hospital, Bergen, Norway1; Section for Microbiology and
Immunology, the Gade Institute, University of Bergen, Bergen, Norway2; Institute of Medicine, University of Bergen,
Bergen, Norway3; and Department of Medicine, Haukeland University Hospital, Bergen, Norway4
Received 15 May 2009/Returned for modification 23 July 2009/Accepted 1 September 2009
RipSeq (iSentio, Bergen, Norway) is a web-based application for the analysis of mixed DNA chromatograms.
It opens the possibility to analyze chromatograms obtained by direct 16S rRNA gene sequencing from
polybacterial human clinical samples. In this study, we used direct 16S rRNA gene sequencing to investigate
264 samples from a wide range of suspected human bacterial infections. The sequence-based identification was
compared with the results from routine culture-based identification. A total of 151 samples were positive by the
first PCR, producing 85 pure and 66 mixed DNA chromatograms. All mixed chromatograms were analyzed by
RipSeq, although seven were so complex that only the dominant bacterial sequences could be identified. In
general, sequence-based identification detected a larger number of species than did culture for samples from
patients who had received antibiotics prior to sample collection and for samples containing anaerobic bacteria.
RipSeq made it possible to apply this supplementary diagnostic tool to typical polybacterial specimens, such
as internal abscesses, pleural fluids, and bile.
Detection and identification of bacteria directly from clinical
samples by broad-range PCR targeting the 16S rRNA gene
and DNA sequencing (direct 16S rRNA gene sequencing)
make up a well-established method in many laboratories. This
method gives the possibility to identify bacteria that died during transportation or as a consequence of antibiotic treatment
and to uncover bacteria with special growth requirements. The
latest advances in PCR and sequencing technology also offer a
more rapid identification than that obtained with standard
phenotypic methods that depend on bacterial growth.
Because of difficulties in the interpretation of DNA chromatograms resulting from direct sequencing of polybacterial
samples, the use of this diagnostic tool has been limited to
infections that are predominantly monobacterial. We earlier
described RipSeq (iSentio, Bergen, Norway), a web-based application for the analysis of mixed DNA chromatograms (12).
In the same article, we presented the RipSeq performance on
a number of mixed DNA chromatograms obtained by direct
sequencing from saline suspensions containing two and three
different bacterial species and discussed the possible benefits
and limitations one could experience if the method was to be
applied to human clinical samples. In this study, direct 16S
rRNA gene sequencing was used to investigate 264 human
clinical samples from a wide range of locations, including typical polybacterial specimens such as abscesses and pleural fluids. All mixed DNA chromatograms were analyzed with the
RipSeq program, and the sequence-based results were com-
pared to routine culture-based diagnostics in our hospital laboratory.
We also discuss special concerns in the selection of a lysis
procedure and primers for use on polymicrobial samples as
well as the establishment of a reliable negative control.
MATERIALS AND METHODS
During five different periods between December 2006 and January 2009,
residual material from 264 clinical samples submitted to our laboratory was
collected. The collection was limited to samples from normally sterile body sites,
and the following specimens were included: abscesses from internal organs/
spaces, soft tissue abscesses and perioperative deep soft tissue material, biopsies
from osteomyelitic lesions (including spondylodiscitis), bile, cerebrospinal fluid,
and cardiovascular samples, in addition to broth from positive mixed blood
cultures. Pleural fluids, peritoneal fluids, synovial fluids, and tissue from around
prosthetic joints were included if the clinical information stated that infection
was suspected or if accompanied with information about elevated infection
parameters, fever, or antibiotic treatment. In addition, for pleural fluids, all
samples from intensive care units and the department of infectious diseases were
accepted independent of clinical information. For liquid samples, a minimum of
200 l of material was required, and for solid tissue, a sample at least the size of a
fingernail was required. No sample was more than 5 days old at the day of
inclusion, but after lysis, samples could be stored at ⫺80°C for several weeks
before further processing. The results from the direct sequencing analysis were
compared to the results obtained by routine culture-based diagnostics in our
laboratory.
Conventional microbiological methods. The routine culture methods included
aerobic culture on blood agar and chocolate agar plates and, when appropriate,
anaerobic culture on a fastidious anaerobic agar plate. Tissues and body fluids
were also cultured in an enrichment broth (brain heart infusion broth). Identification of the isolated colonies was based on routine microbiological methods,
including Vitek II (bioMérieux), API (bioMérieux), Rapid ID 32 A (bioMérieux), and RapID-ANA II (Innovative Diagnostic Systems). If requested by
the medical microbiologist in charge, 16S rRNA gene sequencing from pure
culture was used for identification/confirmation of the phenotypic identification.
Pre-PCR treatment. Between 200 and 800 l of sample material was added to
a bead-containing tube (SeptiFast lysis kit; Roche), together with 400 l of
bacterial lysis buffer (Roche). Eight hundred microliters was the maximum capacity of the bead-containing tube and was used for liquid samples with low
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Haukeland University Hospital, Paradisleitet
12, 5231 Paradis, Bergen, Norway. Phone: 47 48 19 26 19. Fax: 47 94 76
59 36. E-mail: oyvind.kommedal@isentio.com.
䌤
Published ahead of print on 9 September 2009.
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FIG. 1. Direct 16S rRNA gene sequencing versus standard culture techniques for the detection of bacteria in 160 clinical samples. AB⫹,
antibiotic treatment already started at time of sample collection; AB⫺, no antibiotic treatment at time of sample collection; n, number of samples.
viscosity. For other samples, 400 l was used, if available. Two hundred microliters was the smallest volume that would still provide 400 l of supernatant for
the subsequent DNA purification and was the smallest volume accepted for all
specimens. A negative control containing lysis buffer and 400 l of PCR-grade
water was included in every batch of samples. The samples were run twice for 45 s
each in a FastPrep machine (Cepheid) at speed 6.5. After a short spin, 400 l of
supernatant was transferred to a MagNa Pure Compact automated extractor
(Roche), and DNA was extracted and purified using the “total nucleic acid”
program according to the manufacturer’s instructions. Of the resulting 50 l of
eluate, 2 l was used as a template in the broad-range PCR. An amplification
control to detect possible remaining inhibitory substances in the samples was not
included in this assay.
Primers. The following primers were used for the first PCR as well as the cycle
sequencing reactions: forward primer, 5⬘-CGG-CCC-AGA-CTC-CTA-CGG-GAGGCA-GCA-3⬘; and reverse primer, 5⬘-GCG-TGG-ACT-ACC-AGG-GTA-TCTAAT-CC-3⬘.
The PCR product obtained with these primers has a size of approximately 460
bp, covering the variable areas V3 and V4 of the 16S rRNA gene (3). By use of
the RipSeq program, these primers were found to bind more poorly to Chlamydia trachomatis (F4), Chlamydophila abortus and Chlamydophila psittaci (F4,
F9), Chlamydophila pneumoniae (F4), Coxiella burnetii (R4), Dermabacter hominis (R3), Leuconostoc spp. (F4), Microbacterium spp. (R3), and Propionibacterium spp. (R3) (numbers denote the position numbers, from the 3⬘ end, with
mismatches in the forward [F] or reverse [R] primer). No cross-reactivity against
human DNA was seen.
PCR conditions. PCR was performed in a 25-l reaction tube on a SmartCycler real-time apparatus (Cepheid). The PCR mixture consisted of 12.5 l
ExTaq SYBR master mix (TaKaRa, Japan), 0.4 M of each primer, 8.5 l
PCR-grade water, and 2 l extracted DNA. The PCR thermal profile included an
initial polymerase activation step of 10 s at 95°C followed by 40 cycles of 15 s at
95°C, 10 s at 70°C, and 20 s at 72°C.
Positive samples were rerun without SYBR green, using the same protocol as
that described above, but replacing the ExTaq SYBR master mix with ExTaq
Perfect master mix (TaKaRa, Japan). The number of cycles in this PCR was
adjusted based on the results from the SYBR green reaction to make sure that
all positive samples reached the reaction plateau level.
Definition of a positive sample. A positive sample was defined as a sample
reaching the fluorescence threshold value (CT) ⱖ3 cycles before the negative
control did. A sample was also defined as positive if it reached the CT fewer than
three cycles before the negative control if the subsequent melting curve analysis
showed a single distinct peak clearly different from that for the negative control.
Sequencing. The PCR products were spun out of the Smart Cycler reaction
tubes into a 1.5-ml Eppendorf tube and cleaned up using an ExoSAP-IT enzymatic degradation kit (Affymetrix). Sequencing was performed in a core facility
using an ABI Prism 1.1 Big Dye sequencing kit and an ABI 3730 DNA analyzer
(Applied Biosystems).
Interpretation of chromatograms. Mixed DNA chromatograms were analyzed
using the RipSeq web application (iSentio). The RipSeq mixed algorithm
searches against the “16S human pathogen iSentio” database, currently containing about 850 sequences from more than 600 different bacterial species. The
definition of a positive identification with the RipSeq program has been described previously (12). Nonmixed chromatograms were analyzed with both the
RipSeq algorithm and a standard BLAST search against the GenBank database.
Compared to the first 500 bp of the 16S rRNA gene, the area sequenced in this
article has a lower interspecies resolution but also fewer sequence variations
within a given species. For the BLAST search, a similarity of ⬎99.4% (maximum
2 bp difference) together with a ⬎0.4% (minimum 2 bp difference) separation
from other species was considered sufficient for identification to the species level.
Analytical sensitivity of the assay. The PCR was optimized to reach a sensitivity of 1 to 10 genome copies per reaction tube. By spiking of EDTA-blood with
different concentrations of bacteria, using the definition of a positive sample
described above, the sensitivity of the assay was found to be 2,000 to 4,000
genome copies per ml of sample material.
RESULTS
A total of 264 samples were included in the study. Among
these, 13 were abscesses from patients with appendicitis, diverticulitis, or recent colonic surgery located close to or in
direct connection with the colon or appendix. For these samples, sequencing contributed to the detection of a total of 18
anaerobic species not found by culture, but 18 others were
discovered uniquely by culture. Most of the chromatograms
were so complex that only the dominant peaks could be interpreted. Even though they contributed to additional findings,
we concluded that these samples in general were not suitable
for direct sequencing and excluded them from further analysis
and presentations of results.
Of the remaining 251 samples, a total of 160 were positive by
one or both methods. One hundred fifty-one were positive by
the broad-range PCR, and 125 were positive by culture. An
overall comparison of broad-range PCR and culture is shown
in Fig. 1. Eighty-five samples produced pure chromatograms
containing a single bacterial sequence, whereas 66 were mixed
and could not have been interpreted without the RipSeq program. Four samples would have been defined as negative based
on the CT value alone but were included as positive based on
3564
KOMMEDAL ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Overview of positivity rates, antibiotic treatment, and concordance between culture and direct sequencing results for
different specimen categories
Specimen category
No. of
samples
No. of samples positive by
PCR/no. of samples positive
by culture
No. of AB
samplesa
No. of positive samples
with concordanceb
No. of mixed
chromatograms
Abscess—internal organs
Abscess–other abdominal/pelvic
Abscess—retroperitoneal/psoas
Bile
Blood culture bottles
Cardiovascular (various)
Central nervous system (various)
Osteomyelitic lesions
Peritoneal fluids
Pleural fluids
Prosthetic joints
Soft tissue infections
Surgical site infection (incisional)
Surgical site infections (organ/space)
Synovial fluid from native joints
30
6
5
12
10
19
3
20
10
41
17
33
15
21
9
24/15
4/4
5/3
12/12
10/10
9/5
2/0
12/12
3/3
13/8
6/9
24/22
12/10
15/12
0/0
20
5
5
9
0
14
2
3
9
35
4
20
12
15
1
9
3
1
6
7
3
1
8
2
2
5
15
4
3
0
11
3
4
7
10
1
0
1
1
10
0
8
4
6
0
251
151/125
154
69
66
Total
a
b
Number of samples from patients on antibiotic treatment at time of sampling.
Concordance: Number of positive samples with concordance between culture and direct sequencing.
a distinct melting peak, as described in Materials and Methods.
This resulted in the sequence-based detection of Staphylococcus aureus, Staphylococcus epidermidis, Mycobacterium tuberculosis (all confirmed by culture), and Francisella tularensis
(confirmed serologically). An overview of positivity rates, concordances, and percentages of samples affected by antibiotics for
the different categories of specimens is given in Table 1.
Thirty-five of the PCR-positive samples were completely
negative by culture, with 28 presenting pure DNA chromatograms and 7 being mixed. All but one were from patients
already on antibiotic treatment. The exception was an ovarial
abscess containing DNA from Chlamydia trachomatis. Sixtynine samples, 50 monobacterial and 19 polybacterial, gave the
same answer by both culture and direct sequencing. For the
remaining 47 PCR-positive samples, there was partial concordance between culture and sequencing in 37 and no concordance in 10.
For patients not on antibiotic treatment at the time of sample collection, concordance with culture was seen in 41 of 55
PCR-positive samples (75%). For patients who had been on
antibiotic treatment for more than 1 day prior to sample collection, concordant results were found in 28 of 97 PCR-positive samples (29%).
Nine of the PCR-negative samples were positive by culture
and had or might have had clinical relevance. These were two
samples from aorta grafts, growing Propionibacterium acnes
(BH ⫽ growth in enrichment culture only) and one colony of
a Micrococcus sp.; two biopsies from osteomyelitic lesions, with
Staphylococcus aureus and Staphylococcus lugdunensis (BH);
three samples from prosthetic joints, one with Enterococcus
faecalis, one with S. aureus, and one with S. epidermidis (BH);
one spleen bed abscess with P. acnes; and one tissue sample
from an incisional surgical site infection growing P. acnes (BH)
and Streptococcus sanguinis (BH). None of these nine samples
were affected by antibiotics. In eight of the samples, growth
was scarce or intermediate and the number of genome copies
was probably below the analytical sensitivity of the assay. For
the sample from the spleen bed, abscess growth was rich. The
reason for the negative PCR for this sample is most likely the
reverse primer mismatch against P. acnes mentioned earlier.
Previously, the use of 16S rRNA amplification and sequencing
has been limited to specimens expected to be monobacterial. It
has been shown to be useful in the microbiological diagnosis of
brain abscesses (16), endocarditis (13), infected prosthetic joints
(20), meningitis (2, 5, 18), osteomyelitis (9, 21), septic arthritis
(17), spondylodiscitis (8), and vascular graft infections (19). In our
material, samples from these conditions comprised 27% (70 samples) of the total, and 85% of those positive by the broad-range
PCR produced pure chromatograms. The exceptions consisted of
one biopsy from an osteomyelitic lesion (S. aureus and S. epidermidis), one paraprosthetic vascular graft abscess (Corynebacterium tuberculostearicum and Staphylococcus haemolyticus), and
four brain abscesses (Table 2). The largest proportions of mixed
chromatograms were derived from abscesses in internal organs/
spaces, bile, and pleural fluids. These specimens frequently contain anaerobic bacteria, and the majority of patients (74 to 85%)
had been treated with antimicrobial agents prior to sample collection. Concordance between culture and sequencing was low
(Table 1). The total number of positive samples in these three
specimen categories was 66. From these 66 samples, the sum of
bacteria recovered by direct 16S rRNA gene sequencing exclusively was 64 (39 aerobes, 24 anaerobes, and 1 atypical bacterium). For 40 of the 43 samples where sequencing gave additional
information, the patient had already started on antibiotic treatment when the sample was taken. The most interesting findings
were seen among the abscesses and pleural fluids. The detailed
results for these samples are given in Tables 2 and 3.
In addition to a pelvic abscess with Chlamydia trachomatis,
two culture-negative samples contained DNAs from atypical
bacteria. One was a sternum biopsy from a child with acute
lymphatic leukemia and suspected osteomyelitis, from which
abundant DNA from Mycoplasma hominis was isolated (be-
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16S SEQUENCING FROM POLYBACTERIAL CLINICAL SAMPLES
3565
TABLE 2. Overview of results for PCR- and/or culture-positive abscesses in internal organs/spacesd
Abscess location
Sample
IDa
Culture result
Sequencing result
Antimicrobial
treatmentc
Aorta
1A
No growth
Streptococcus pneumoniae/pseudopneumoniaeb
DX
Brain
Brain
Brain
Brain
Brain
2A
3A
4A
5A
6A
7A
8A
Brain
9A
Streptococcus intermedius
Aggregatibacter aphrophilus, Streptococcus intermedius
Streptococcus intermedius
Streptococcus pyogenes
Fusobacteriumnucleatum, Parvimonas micra,
Streptococcus intermedius
Streptococcus intermedius
Campylobacter gracilis, Fusobacterium nucleatum,
Parvimonas micra
Fusobacterium nucleatum
⫺
CX, MZ
⫺
PC, RI
CX, MZ
Brain
Brain
Brain
10A
Streptococcus intermedius
Streptococcus intermedius
Streptococcus milleri group
No growth
Peptostreptococcus sp., Streptococcus
milleri group
No growth
Actinomyces meyeri (Seq), Fusobacterium
nucleatum
Enterococcus sp., Fusobacterium
nucleatum, Peptostreptococcus sp.
No growth
Campylobacter gracilis, Fusobacterium
nucleatum/naviforme, Streptococcus intermedius
CT, MZ
Kidney
Kidney
11A
12A
Escherichia coli
Escherichia coli
Escherichia coli/Shigella spp.
Escherichia coli/Shigella spp.
AM, CI
AM, GE
Liver
Liver
Liver
13A
14A
15A
Clostridium perfringens, Escherichia coli/Shigella spp.
Citrobacter gillenii
Enterococcus durans/faecium, Escherichia
coli/Shigella spp.
CT
Liver
16A*
No growth
Citrobacter yougae
Enterococcus faecium, Escherichia coli,
Klebsiella sp., Staphylococcus
haemolyticus
Staphylococcus haemolyticus
Enterococcus caccae, Gemella hemolysans,
Streptococcus mitis group
PT
CF, CL
MZ, PC
⫺
⫺
⫺
AM
Lung
17A
Lactobacillus sp.
Lactobacillus gassi
Ovary
Ovary
18A
19A
Fusobacterium sp., Peptostreptococcus sp.
No growth
Fusobacterium naviforme, Parvimonas micra
Chlamydia trachomatis
Pancreas
Pancreas
Pancreas
Pancreas
20A
21A
22A
23A
Bacteroides fragilis group, Enterococcus sp.
Mycobacterium tuberculosis
No growth
Coagulase-negative staphylococci
Bacteroides fragilis, Enterococcus durans/faecium
Mycobacterium tuberculosis complex
Neisseria subflava
Campylobacter concisus/mucosalis, Prevotella
melaninogenica/histicola, Staphylococcus capitis/
caprae/epidermidis
Spleen
24A
Enterococcus faecalis, Streptococcus anginosus,
Lactococcus lactis
Fusobacterium nucleatum
PC, MP
⫺
⫺
IP
⫺
PT
IP
Spleen
25A
Enterococcus faecalis, Staphylococcus
epidermidis
No growth
Pelvic
26A
Enterococcus faecalis, Escherichia coli
Enterococcus faecalis, Escherichia coli
CL, MZ
Psoas
Psoas
Psoas
27A
28A
29A
No growth
No growth
Staphylococcus aureus
Enterobacter hormaechei, Enterococcus durans/faecium
Enterococcus durans/faecium, Klebsiella pneumoniae
Staphylococcus aureus
VA, MZ, CI
MP
OX, CL
Retroperitoneal
Retroperitoneal
30A
31A*
Enterococcus faecium
Bacteroides fragilis group, Enterococcus
faecium
Enterococcus durans/faecium, Klebsiella pneumoniae
Bacillus subtilis, Enterococcus sp.
MP
CI, MX, LZ
Subphrenic
32A
Enterococcus durans
CI, MX, LZ
Subphrenic
Subphrenic
33A
34A
Enterococcus sp.
Enterococcus faecium, Klebsiella sp.
Enterococcus durans/faecium, Streptococcus salivarius/
thermophilus
Enterococcus faecalis
Enterococcus durans/faecium, Klebsiella pneumoniae
SSI abdominal (Billroth I)
SSI spleen bed (splenectomy)
SSI kidney bed (nephrectomy)
SSI liver (liver resection)
SSI liver (liver resection)
SSI liver (liver resection)
SSI retroperitoneal (Whipples)
SSI retroperitoneal (Whipples)
SSI subphrenic (ventricular
resection)
SSI subphrenic (unknown)
35A
36A
37A
38A
39A
40A
41A
42A
43A
No growth
Propionibacterium acnes
Citrobacter koseri
Klebsiella pneumoniae
No growth
No growth
Enterococcus sp.
Enterococcus sp.
Coagulase-negative staphylococci, Serratia
sp., Stenotrophomonas maltophilia
Pseudomonas aeruginosa
Haemophilus haemolyticus
Negative
Citrobacter koseri
Haemophilus parainfluenzae, Klebsiella pneumoniae
Staphylococcus capitis/caprae/epidermidis
Propionibacterium acnes
Haemophilus parainfluenzae, Streptococcus agalactiae
Enterococcus durans/faecium, Streptococcus agalactiae
Enterococcus sp., Staphylococcus sp.,
Stenotrophomonas maltophilia
Pseudomonas aeruginosa
44A
⫺
CT, MZ
GE, MZ
CT, MZ
⫺
⫺
⫺
CT
CT, MZ
CT, MZ
CT, MZ
MP
⫺
a
ⴱ, chromatogram was too complex to allow for complete analysis. Only dominant peaks were included.
Separation from several other members of the Streptococcus mitis group by only 1 bp (⬍0.3%).
⫺, no treatment; AM, amoxicillin; CI, ciprofloxacin; CF, cefuroxime; CT, cefotaxime; CX, ceftriaxone; DX, doxycycline; GE, gentamicin; IP, imipenem; LZ,
linezolid; MP, meropenem; MZ, metronidazole; OX, oxacillin; PC, penicillin G; PT, piperacillin-tazobactam; RI, rifampin; VA, vancomycin.
d
Seq, identification was confirmed/performed by 16S rRNA gene sequencing from pure culture; SSI, surgical site infection (with procedure in parenthesis). Results
in bold indicate discordant findings.
b
c
coming positive 12 cycles before the negative control did). The
other was from a young male without known immunodeficiency who was diagnosed with a low-grade spondylodiscitis.
The PCR became positive four cycles before the negative con-
trol did and showed a distinct peak in the melting curve analysis. The resulting pure chromatogram gave a 99.9% match
against “Flexispira rappini.” Other unusual findings comprised
a nonmixed DNA chromatogram from a subhepatic abscess
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KOMMEDAL ET AL.
J. CLIN. MICROBIOL.
TABLE 3. Overview of results for PCR- and/or culture-positive pleural fluidsa
Sample
ID
Culture result
1P
Streptococcus intermedius
2P*
Coagulase-negative staphylococci, diphtheroids,
Streptococcus constellatus
Anaerobic gram-positive rod, Prevotella sp.
Streptococcus intermedius
3P
4P
5P
6P
7P
8P*
9P
10P
Escherichia coli
No growth
No growth
Campylobacter gracilis (Seq), Eikenella corrodens
(Seq), Streptococcus parasanguinis
No growth
No growth
11P*
Neisseria sp., Streptococcus sp.
12P
Prevotella sp., Streptococcus mitis group
13P
No growth
a
Antimicrobial
treatment
Sequencing result
Campylobacter gracilis, Fusobacterium nucleatum/
canifelinum, Streptococcus
intermedius/anginosus
Campylobacter gracilis, Fusobacterium nucleatum
Dialister pneumosintes, Peptostreptococcus stomatis
Fusobacterium naviforme, Parvimonas micra,
Streptococcus intermedius
Escherichia coli/Shigella spp.
Neisseria pharyngis, Streptococcus sp.
Clostridium tertium, Enterococcus faecalis
Campylobacter gracilis, Fusobacterium nucleatum,
Parvimonas micra, Prevotella pleuritidis
Streptococcus pyogenes
Parvimonas micra, Peptostreptococcus stomatis,
Streptococcus anginosus
Granulicatella adjacens/para-adiacens,
Streptococcus mitis group
Fusobacterium nucleatum, Prevotella histicola,
Streptococcus constellatus
Streptococcus intermedius
CT, GE, PC
CI, CL
CT
PC, CT
CI, MP
CT, MZ
PT
PC
PC, CT
PT
CT
PC, GA
CT, VA
See the footnotes to Table 2 for further explanations.
with 100% similarity to the type strain of Haemophilus haemolyticus (EU909671) and the discovery of Campylobacter spp. in
six polymicrobial samples (two brain abscesses, one pancreatic
abscess, and three pleural fluids), among which only one was
also found by culture.
The results for the positive blood culture bottles are listed
in Table 4. These are representatives of samples where the
comparison between culture-based isolation and sequencebased detection was not biased by antibiotic treatment or
sample collection and transportation procedures. Still, the
results were not in concordance for 3 of 10 samples (samples
2, 4, and 8).
DISCUSSION
The main purpose of this article was to investigate the usefulness of mixed DNA chromatogram analysis of human clinical specimens. The majority of polybacterial samples were
from various abscesses, pleural fluids, and bile, but occasional
mixed chromatograms were also derived from typically
monobacterial specimens. Many bacteria were recovered exclusively by sequencing, especially for patients who had been
treated with antibiotics prior to sample collection. Because our
hospital does not utilize dedicated transport containers for
anaerobic bacteria, additional anaerobic species were found by
sequencing in samples from both antibiotic-treated and untreated patients.
For all specimen categories, most bacteria found are widely
accepted to have a pathogenic potential in humans, but a small
selection mentioned in Results are more unusual and have a
less definite status. Mycoplasma hominis has previously been
reported as the probable cause of a range of extragenital infections. An overview with a focus on sternal infections and
mediastinitis is given by Mattila et al. (14). Fewer reports exist
of human infections with “Flexispira rappini”-like organisms,
and there are still unsolved matters concerning their taxonomy.
The reference that gave the closest match with our sequence
TABLE 4. Comparison between culture and direct sequencing results for positive blood culture bottlesa
Sample
ID
Culture result
Sequencing result
3
Bacteroides fragilis group, Clostridium boltae (Seq)
Bacteroides fragilis group, Eubacterium lentum (Seq), Pseudomonas
aeruginosa, Sutterella wadsworthensis (Seq)
Bacteroides fragilis group, Clostridium boltae (Seq), Peptostreptococcus sp.
4
Parvimonas micra (Seq), Dialister pneumosintes (Seq)
5
6
7
8
9
10
Enterococcus casseliflavus, Escherichia coli
Escherichia coli, Streptococcus galactolyticus
Escherichia coli, Streptococcus agalactiae
Bacteroides fragilis group, Clostridium sp., Escherichia coli
Escherichia coli, Pseudomonas aeruginosa
Enterococcus faecalis, Staphylococcus hominis
Bacteroides fragilis, Clostridium boltae
Bacteroides ovatus/dorei, Dialister pigra, Sutterella
wadsworthensis
Bacteroides fragilis, Clostridium boltae, Ruminococcus
gnavus
Catonella sp., Dialister pneumosintes, Parvimonas micra,
Porphyromonas asaccharolytica
Enterococcus casseliflavus/gallinarum, Escherichia coli
Escherichia coli, Streptococcus galactolyticus
Escherichia coli, Streptococcus agalactiae
Clostridium ramosum, Escherichia coli
Escherichia coli, Pseudomonas aeruginosa
Enterococcus faecalis, Staphylococcus homins/lugdunensis
1
2
a
See the footnotes to Table 2 for further explanations.
VOL. 47, 2009
16S SEQUENCING FROM POLYBACTERIAL CLINICAL SAMPLES
was a blood culture isolate from a patient with diarrhea
(GenBank accession no. AF286053). Two case reports of bone
infections with “Flexispira rappini”-like bacteria have been published (4, 9). Our patient recovered on treatment with doxycycline, gentamicin, and penicillin G. H. haemolyticus is part of
the normal flora of the upper respiratory tract and is considered to be a nonpathogen (15). Our sample was taken from a
subhepatic abscess in a 67-year-old woman 9 days after a Billroth I operation. She presented with fever and an elevated
C-reactive protein level and neutrophil count and had been
treated with antibiotics for 4 days before specimen collection.
She recovered upon abscess drainage and treatment with cefotaxime. A review of the literature confirms the role of
Campylobacter gracilis in certain extra-oro-intestinal abscesses
and empyemata (6, 11). Most recently, it was discovered by
broad-range amplification and cloning in three polybacterial
brain abscesses (1).
The blood culture results were included mainly to get a
comparison between culture and sequencing, unaffected by
antibiotics, sample collection procedures, or transportation.
Some of the cultured bacteria were not found by sequencing.
These are examples of one of the limitations attached to sequencing directly from mixed clinical samples. Because all bacteria in a sample will be competing for the same reagents, those
present at the lowest concentrations might be outcompeted in
the PCR and not visible in the resulting DNA chromatogram.
We have shown experimentally that this is likely to occur when
the molar ratio exceeds 1:10. In two of the blood cultures, some
of the anaerobic bacteria were detected solely by sequencing.
This may be due to bacterial lysis in the bottles, unsuccessful
subcultivation, or difficulties in differentiating between colonies on the agar plates.
The RipSeq algorithm has been validated for samples containing up to three different species of bacteria. Occasionally,
four bacterial species can be accepted, e.g., if variations in
primer affinities lead to the detection of different bacteria with
the forward and reverse chromatograms. For two abscesses,
two bile samples, and three pleural fluids, the chromatograms
were so complex that all peaks could not be included without
exceeding this limitation of the RipSeq algorithm (Tables 2
and 3). A high y-axis cutoff was used to include only the most
dominant peaks and to make the RipSeq analysis valid. All
seven samples had been exposed to antibiotics, and despite
ignoring the lower portions of the chromatograms, additional
bacteria not found by culture were still detected in all of them.
The limitation in how complex a chromatogram can be before specificity becomes too low and the competition for reagents in mixed samples are the two major challenges for the
use of broad-range PCR and DNA sequencing directly from
polybacterial samples. This was clearly illustrated with abscesses originating from the colon and appendix, but also with
the other samples where a proportion of the involved bacteria
were found by culture only. One possible way to reduce these
problems is to use Gram stain type-specific broad-range primers and to amplify gram-positive and gram-negative bacteria in
different tubes. In a recent publication, these problems were
omitted by the use of cloning and high-throughput pyrosequencing in the investigation of brain abscesses (1). A surprising level of complexity was demonstrated in some of the samples. Unfortunately, this sort of diagnostics is currently not
3567
within reach for the routine laboratory. A less costly alternative
is the use of denaturing gradient gel electrophoresis followed
by DNA sequencing of the different fragments. This approach
is labor-intensive and technically challenging, and although it
will not in theory have a limitation when it comes to the
number of different species in a sample, it will have shortcomings in detecting the minor populations in samples with large
differences in the relative concentrations of the different participants. In addition to denaturing gradient gel electrophoresis, a number of methods have been developed that work by the
principle of separating the different DNA fragments based on
their physical properties. The WAVE system (Transgenomic,
Omaha, NE) uses denaturing high-performance liquid chromatography to separate the different DNA fragments in combination with an automated fragment collector and has been
shown to be useful in the analysis of complex human clinical
samples (7). The automatic fragment collection makes it easier
to further analyze the fragments with DNA sequencing. A
proportion of the fragments will not be separable by denaturing high-performance liquid chromatography, but DNA chromatograms for these will typically contain only two or three
different species and should be suitable for RipSeq analysis.
The limited sensitivity is the main challenge for direct 16S
rRNA gene sequencing in general (10). For our assay, we
found a sensitivity of 2,000 to 4,000 genome copies per ml of
sample material. Based on the chosen pre-PCR treatment, the
amount of template used in the PCR, and the definition of a
positive sample, this is very close to the theoretical detection
limit. The consequence of a low sensitivity is that a negative
broad-range PCR can never exclude the presence of bacterial
DNA in a sample, and the laboratory should always make this
clear to the physician in charge of the patient. In addition, in
this study, because we did not include an amplification control,
some of the investigated samples might have produced falsenegative results due to unrecognized inhibitory substances.
The choice of the most relevant specimens could be optimized further. As already mentioned, sequencing from abscesses in direct connection to the colon/appendix was judged
not to be clinically useful. Peritoneal fluids had a low positivity
rate, and sequencing gave little added value. Sequencing from
osteomyelitic lesions in adults and from prosthetic joints gave
additional information for a number of samples where the
patients had been treated with antibiotics but had a lower
sensitivity than culture with nontreated samples. The inclusion
criteria used for pleural fluids led to the investigation of a high
proportion of negative samples. Retrospectively, we found that
a pulmonary infection was suspected in about 60% of the cases,
including all of the positive cases. The remaining 40% were
chiefly thought to be caused by cardiovascular conditions or
cancer. If we had access to this information in the first place,
the positivity rate would have improved from 30 to 48%.
The use of real-time PCR with SYBR green detection has
important advantages compared to traditional PCR with gelbased detection. With gel-based detection, the number of cycles in the first PCR has to be limited to ensure that no visible
band appears in the negative control. The amplification process with positive samples containing lower levels of DNA will
then be aborted before it reaches the plateau level, resulting in
large intersample variations in the amount of DNA used in the
cycle sequencing reactions. With real-time PCR, all samples
3568
KOMMEDAL ET AL.
can be run to the plateau level, since a positive sample is
defined relative to when the negative control reaches its CT
value. More importantly, it gives a semiquantitative measure of
how much bacterial DNA a sample contains. A sample that
reaches its CT value early contains more bacterial DNA, and
the final result is less likely to represent contamination than if
it was from a sample that reaches its CT value just before the
cutoff, even if both are positive by definition. Finally, we found
that with the possibility of doing mixed chromatogram analysis,
the definition of a positive sample had to be modified. For
some of the samples becoming positive three to six cycles
before the negative control did, contaminant bacterial DNAs
from the reagents were still detectable as lower peaks in the
chromatograms. Consequently, only the dominant peaks were
considered to be relevant, and these samples were analyzed
using a higher cutoff on the y axis in the RipSeq program.
When DNAs from multiple species are going to compete for
the same primers, the primers not only must be universal but
also must bind equally efficiently to the respective bacteria. A
bacterium with a single mismatch close to the 3⬘ end of one of
the primers will be outcompeted in the amplification process
by a bacterium with a perfect match. It is also crucial that the
lysis procedure is equally efficient for all relevant bacteria. If
these two matters are not addressed properly, the DNA chromatograms may give a false impression of the number of bacteria in the sample (12).
The single strongest reason to consider direct 16S rRNA
gene sequencing as a supplement to culture should be the
administration of antibiotics prior to sample collection. In hospitals that do not have dedicated transport systems for anaerobic bacteria, it should also be considered for important samples such as brain abscesses, regardless of antimicrobial status.
There has been a tendency in our hospital to await culture
results and eventually to proceed to direct sequencing only
with culture-negative samples. This policy fails to take into
consideration that in samples affected by antibiotics some species can still be able to grow, whereas others are not. An
incomplete microbiological answer can be misleading and result in insufficient antimicrobial coverage, especially in patients
where standard empirical therapy cannot be used or in patients
that are transferred to oral treatment. This article shows that
with the RipSeq program, the use of direct 16S rRNA gene
sequencing can be expanded to a number of typical polymicrobial specimens. It was found to be of particular value for
internal abscesses, pleural fluids, and bile. The program allowed for partial or complete interpretation of all 66 mixed
DNA chromatograms in the study.
ACKNOWLEDGMENTS
This work was supported by The Research Council of Norway and
Innovation Norway.
A patent application has been filed for several aspects of the RipSeq
algorithm. The RipSeq program is accessible through a commercial
web service owned by iSentio AS. The first author is a coowner and the
CMO of this company.
J. CLIN. MICROBIOL.
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