Published online: 2018-12-11

108 Original Article

Bacterial Profile of Middle Ear Fluid with Recurrent

Acute Otitis Media Infection Using Culture
Independent 16S rDNA Gene Sequencing
Geetha Nagaraj1 Anurag Girdhar2 Jagdish Chinnappa2 Feroze Ganaie1 Vandana Govindan1
Kadahalli Lingegowda Ravikumar1

1 Central Research Laboratory, Department of Microbiology, Kempegowda
Institute of Medical Sciences, Bengaluru, Karnataka, India
2 Department of Pediatrics, Manipal Hospital, Bengaluru, Karnataka, India
J Pediatr Infect Dis 2019;14:108–115.
Address for correspondence Geetha Nagaraj, MSc, Central Research
Laboratory, Department of Microbiology, Kempegowda Institute of
Medical Sciences, Hospital and Research Centre, K. R. Road, V. V.
Puram, Bengaluru 560 004, Karnataka, India
(e-mail: geetha.ndri@gmail.com).

Abstract Background Recurrent otitis media is one of the common infections of childhood.
The causative bacterial pathogen is one of the major risk factors of recurrent infection.

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Keywords
► recurrent acute otitis
media
► bacterial etiology
► 16s rDNA sequencing
With limited availability of Indian data, we performed this study to identify the bacterial
pathogens.
Materials and Methods Otitis media cases were diagnosed based on clinical criteria.
Thirty-six middle ear fluid (MEF) samples were collected by tympanocentesis and
cultured for pathogens. Seventy-eight per cent of the cases had three previous
episodes of otitis media in the past 6 months; the remaining 22% had four episodes
in the preceding 6 months. At the time of sample collection, all patients were on
antibiotic coverage. Genomic DNA was extracted from MEF samples using Qiagen DNA
mini Kit. The 16s rDNA polymerase chain reaction (PCR) and quantitative multiplex
(qmPCR) for Streptococcus pneumoniae was performed on these samples. Streptococcus
pneumoniae–positive samples were serotyped using PCRSeqTyping.
Results None of the 36 samples showed growth by conventional culture. The 16s
rDNA PCR identified bacterial pathogens in 33 samples. Four samples gave mixed
reads. The organisms identified were Neisseria spp. other than Neisseria meningitidis
(n ¼ 7), N. meningitidis (n ¼ 8), Lactococcus spp. (n ¼ 5), S. pneumoniae (n ¼ 2),
Pseudomonas aeruginosa (n ¼ 2), Haemophilus influenzae (n ¼ 1), Salmonella infantis
(n ¼ 1), Staphylococcus epidermidis (n ¼ 1), Staphylococcus auricularis (n ¼ 1), and
Streptococcus sp. (n ¼ 1). The qmPCR detected the presence of S. pneumoniae in six
samples. PCRSeqTyping was able to identify Serotype 19A in two samples positive for S.
pneumoniae.
Conclusion The study demonstrates the usefulness of 16s rDNA PCR protocol to
identify the bacterial pathogens in MEF by a culture-independent method. Neisseria
spp. were the predominant species identified followed by Lactococcus spp. and S.
pneumoniae. Detection of pneumococci by 16s rDNA PCR correlated well with qmPCR-
based detection and PCRSeqTyping.

received Copyright © 2019 by Georg Thieme DOI https://doi.org/

May 7, 2018 Verlag KG, Stuttgart · New York 10.1055/s-0038-1675786.
accepted after revision ISSN 1305-7707.
September 28, 2018
published online
December 11, 2018

Introduction
Acute otitis media (AOM) is a very common pediatric infection
and a leading cause of health care visits and antibiotic pre-
scription.1 Recurrent acute otitis media (rAOM) is encountered
in a subpopulation of 5 to 30% of all children with AOM.2
Traditionally, the initial AOM attack occurs at an early age.
Comorbid airway infection, sibling AOM history, bottle feed-
ing, daycare condition, genetic predisposition, premature
birth, male sex, and some ethnicities have been considered
risk factors for rAOM. If this condition is left untreated, it may
lead to middle ear damage followed by various complications
such as otorrhea, mastoiditis, facial nerve paralysis, intracra-
nial abscess, meningitis, etc.3 Due to the low socioeconomic
status, overcrowding, poor hygiene, inadequate health care,
and recurrent upper respiratory tract infection, the burden is
high in low- and middle-income countries.4
The organism causing otitis media and its resistance to the
conventional antibiotics are important factors in determining
whether otitis media will follow a recurrent course. Hence, it
becomes important to determine the causative organism in
individual cases to better understand the propensity for
different bacteria to follow a recurrent course. The microbiol-
ogy of rAOM is often more complex than in isolated episodes of
AOM, but the most common bacteria nevertheless remain
Streptococcus pneumoniae, Haemophilus influenzae, and Mor-
axella catarrhalis with these bacteria accounting for 80% of all
cases. The most frequent S. pneumoniae serotypes involved are
19F, 23F, 14, 6B, 6A, 19A, 9V, 3; 18.1% of H. influenzae belong to
serotype b, the remaining usually being nontypeable strains.
The frequency of mixed cultures is variable but they certainly
exist. Fifteen to 34% of middle ear effusions, obtained from
patients with AOM, are culture negative. Nonviable bacteria,
viruses, chlamydiae, mycoplasmas, and anaerobes may
account for some of these cases.5–8
Our understanding of the microbiology of the middle ear in
healthy and diseased states has been largely derived from
bacterial culture studies. However, culture results are influ-
enced by factors such as prior antibiotic treatment, sampling,
and processing methods. In contrast, culture-independent
molecular techniques can provide a more accurate assessment
of the microbial communities growing in clinical samples. PCRs
have revolutionized the detection of bacteria in clinical samples
since their widespread introduction in the 1990s. Although
qPCR is by far the most frequently used molecular technique in
microbial diagnostics, in certain scenarios, a broad-range 16S
rDNA (ribosomal DNA) PCR is increasingly used.9 The technol-
ogy is mainly used to detect and identify bacterial pathogens in
clinical specimens that are culture negative.10–14 The techni-
ques has been applied on various samples such as tissue, pus,
blood, cerebrospinal fluid, and other body fluids15–22 to identify
the causative bacteria. Broad-range 16S rDNA gene PCR is
particularly suitable for bacteria that are difficult to culture
such as Mycobacterium genavense,23 Tropheryma whipplei,24
Ehrlichia chaffeensis,25 and Coxiella burnetii.26
Here, we aimed to study the bacterial etiology of middle
ear fluid (MEF) collected from children with rAOM, using
culture-independent 16S rDNA gene sequencing.
Bacterial Profile of Middle Ear Fluid Nagaraj et al. 109
Materials and Methods
Study Design and Population
The study was conducted in two tertiary-care teaching hospi-
tals in Bangalore. Thirty-six pediatric patients aged 6 months
to 5 years presenting to the pediatric outpatient department
with recurrent otitis media undergoing tympanocentesis were
enrolled. Clinically confirmed cases of rAOM as per American
Academy of Pediatrics criteria were recruited for the study.
Informed consent was obtained from each parent/guardian
prior to performing study-specific procedure. The protocol
was reviewed and approved by local ethics committees, and
the study was conducted according to Good Clinical Practice
and the declaration of Helsinki.
Study Procedures
Children with rAOM presenting to study physicians were
anonymously recorded in screening logbooks and assessed
for eligibility. Only those cases that were undergoing tympa-
nocentesis as advised by the clinician were included. Demo-
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graphic characteristics, number of previous episodes of AOM,
prior antibiotic use for the current episode, and general
symptoms of eligible children were collected. Clinical exam-
inations, including assessments of tympanic membrane bulge
and redness, were performed at baseline (►Table 1).
Sample Collection, Handling, and Transport
MEF samples were collected by tympanocentesis. Tympano-
centesis was performed at enrollment by an otolaryngolo-
gist, as described elsewhere.27 The collected MEF samples
were transported to microbiology laboratory within 1 hour
in an ice box.
Bacterial Culture
MEF samples were directly inoculated onto 5% sheep blood
agar, chocolate agar, and MacConkey agar (HiMedia, India).
The MacConkey agar plates were incubated aerobically, while
chocolate and blood agar plates were incubated for 24 to
48 hours at 37°C in 5% carbon dioxide. The plates were
observed for the colony characters of bacterium, Gram stain-
ing, and biochemical reaction using the standard methods.28
Molecular Analysis
DNA Extraction
DNA extraction from the MEF samples was done using
QIAamp DNA Mini Kit with automated DNA extractor, QIA-
cube (Qiagen, Germany), as per manufacturer’s protocol.
Quantity and quality of the extracted DNA were measured
spectrophotometrically at 260 nm absorbance with Nano-
drop 2000 (Thermo Fisher Scientific, United States).
Real-Time Polymerase Chain Reaction of Clinical Samples
The detection of S. pneumoniae DNA in the MEF specimens
was performed for LytA, Ply, psaA, and spn9802 genes in
quantitative multiplex real-time PCR (RT-PCR) assays. DNA
extracted (5 µL of undiluted DNA) from MEF specimens were
used in the amplification reactions. All assays with each
Journal of Pediatric Infectious Diseases Vol. 14 No. 3/2019
110 Bacterial Profile of Middle Ear Fluid Nagaraj et al.

Table 1 Demographic and clinical details for the 36 patients from whom middle ear fluids were obtained

Sl.no. Sample ID Gender Age (mo) Fever TM TM Number of Prior antibiotic use
bulge redness previous episodes for current episode
1 2024513 M 50 Present Present Present 3 No
2 1126676 M 54 Present Present Present 3 No
3 2023493 M 44 Present Present Present 4 No
4 931997 M 32 Present Present Present 3 No
5 1652702 M 48 Present Present Absent 3 Yes
6 1847515 F 40 Present Present Present 4 No
7 1400867 F 38 Present Present Present 3 No
8 2031074 M 36 Present Present Present 3 No
9 2046322 M 54 Present Present Present 3 No
10 1855960 M 42 Present Present Present 3 No
11 2048502 M 46 Present Present Present 3 Yes
12 1408423 M 42 Present Present Present 4 No
13 1111215 M 55 Present Present Present 3 Yes

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14 AK F 13 Present Present Present 3 No
15 2073167 M 22 Present Present Present 3 Yes
16 823627 M 12 Present Present Absent 3 No
17 2051377 M 14 Present Present Present 3 No
18 742455 M 16 Present Present Present 3 No
19 2048121 M 48 Present Present Absent 3 Yes
20 2042520 M 17 Present Present Present 3 Yes
21 1975697 M 52 Present Present Present 3 Yes
22 162156 M 25 Present Present Absent 4 Yes
23 171412 M 15 Present Present Present 3 No
24 821450 M 16 Present Present Present 3 Yes
25 192541 M 14 Present Present Present 3 No
26 245068 M 12 Present Present Present 3 No
27 174390 F 14 Present Present Present 3 No
28 184732 M 35 Present Present Absent 4 No
29 180156 M 22 Present Present Present 4 No
30 2012121 M 13 Present Present Present 3 No
31 1981901 M 19 Present Present Present 3 No
32 2014379 M 16 Present Present Present 3 Yes
33 1814067 M 18 Present Present Present 3 No
34 2049143 M 12 Present Present Absent 3 No
35 198479 M 11 Present Present Present 4 Yes
36 2024123 M 16 Present Present Present 3 Yes

clinical sample were performed in duplicate. A specimen was
considered positive if three of the four genes gave a positive
result within the 35-cycle cutoff. qmPCR assay was per-
formed as described in detail in Ganaie et al.29 A control
reaction with GAPDH human gene was performed indepen-
dently with each sample to check for the presence of
inhibitors.30 The primer and probe sequences are listed
in ►Table 2.
Serotyping of Streptococcus pneumoniae by PCRSeqTyping
Streptococcus pneumoniae–positive samples were subjected
for serotyping by PCR amplification and sequencing of cpsB
region as detailed in Nagaraj et al.31 Briefly, PCR amplifica-
tion and sequencing was performed using cpsB primers.32,33
The sequence data were used to interrogate the GenBank
database (http://www.ncbi.nlm.nih.gov/blast/) and assigned
to serotype using the criteria as per protocol.32 Serotype of

Journal of Pediatric Infectious Diseases Vol. 14 No. 3/2019

 Bacterial Profile of Middle Ear Fluid Nagaraj et al. 111

Table 2 Results of 16s rDNA sequencing and qmPCR and serotyping for Streptococcus pneumoniae on 36 middle ear fluid samples

Sl.no. Sample Number of qmPCR for Average Average Organism identified by Serotype—PCRSeqTyping
ID previous Streptococcus copy Ct value 16s rDNA sequencing
episodes Pneumoniae number
1 2024513 3 Positive 436 21.38 Streptococcus pneumoniae 19A
2 1126676 3 Negative Lactococcus sp.
3 2023493 4 Negative No data
4 931997 3 Negative Lactococcus sp.
5 1652702 3 Negative Lactococcus sp.
6 1847515 4 Negative Lactococcus sp.
7 1400867 3 Negative Lactococcus sp.
8 2031074 3 Negative Neisseria meningitidis
9 2046322 3 Negative No data
10 1855960 3 Positive 5 28.72 Mixed reads during Amplification of cpsB was not
sequencing. Single observed due to low copy
organism was not number
identified
11 2048502 3 Negative Staphylococcus epidermidis
12 1408423 4 Negative Streptococcus sp.

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13 1111215 3 Negative Neisseria meningitidis
14 AK 3 Negative Haemophilus influenzae
15 2073167 3 Negative No data
16 823627 3 Negative Neisseria oralis
17 2051377 3 Negative Staphylococcus auricularis
18 742455 3 Negative Neisseria sp.
19 2048121 3 Negative Neisseria meningitidis
20 2042520 3 Negative Neisseria Mucosa
21 1975697 3 Negative Neisseria sp.
22 162156 4 Negative Neisseria sp.
23 171412 3 Positive 16 30.17 Mixed reads during Amplification of cpsB was not
sequencing. Single observed due to low copy
organism was not number
identified
24 821450 3 Negative Neisseria sp.
25 192541 3 Negative Neisseria Meningitidis
26 245068 3 Negative Neisseria Meningitidis
27 174390 3 Negative Pseudomonas aeruginosa
28 184732 4 Negative Salmonella infantis
29 180156 4 Negative Pseudomonas aeruginosa
30 2012121 3 Negative Neisseria meningitidis
31 1981901 3 Positive 253 25.52 Streptococcus pneumoniae 19A
32 2014379 3 Positive 15 29.23 Mixed reads during Amplification of cpsB was not
sequencing. Single observed due to low copy
organism was not number
identified
33 1814067 3 Positive 4 28.48 Mixed reads during Amplification of cpsB was not
sequencing. Single observed due to low copy
organism was not number
identified
34 2049143 3 Negative Neisseria meningitidis
35 198479 4 Negative Neisseria meningitidis
36 2024123 3 Negative Neisseria sp.

Journal of Pediatric Infectious Diseases Vol. 14 No. 3/2019

112 Bacterial Profile of Middle Ear Fluid Nagaraj et al.
the cpsB nucleotide sequence from GenBank with the highest
BLAST bit score was assigned, provided that sequence iden-
tity was >99% with the query amplicon nucleotide sequence.
16s rDNA Polymerase Chain Reaction and Sequencing
PCR reaction was performed using the primers 16s-FP: 5′-
AGAGTTTGATCMTGGCTCAG-3′ and 16s-RP: 5′-TACGGY-
TACCTTGTTACGACTT-3′.8 The reaction mixture contained
10 ng of genomic DNA, 0.75 units XT-5 polymerase (3 U/
μL) (Merck), 1X XT5A-assay buffer (10X), 1 μL deoxynucleo-
side triphosphates (dNTPs, 2.5 mM each, Fermentas), 100 ng
of each primer and made up to a final volume of 25 μL with
DNase/DNAse-free distilled water (Gibco).
Thermal cycling was performed in the GeneAmp PCR
system 9700 (Applied Biosystems) with conditions: 94°C
for 5 minutes, followed by 35 amplification cycles of 94°C
for 30 seconds, 55°C for 30 seconds, 72°C for 90 seconds, and
final extension at 72°C for 5 minutes. The PCR products were
separated by electrophoresis on 1.0% agarose gel for 45 min-
utes at 80 V in 1X Tris-acetate EDTA buffer. Ethidium bro-
mide-stained DNA products were visualized under
ultraviolet illumination and sized by using a 1-kb DNA
molecular size marker (Fermentas).
Sequencing and Data Analysis
PCR products were purified using Qiagen PCR purification kit
following manufacturer’s protocol. The purified PCR pro-
ducts were subjected to sequencing employing the BigDye
Sequence Terminator kit V3.1 (Applied Biosystems) and
analyzed on ABI 3730 XL Genetic Analyzer (Applied Biosys-
tems). Sequencing was performed in both directions using
forward and reverse primer. DNA sequences obtained were
used to interrogate the GenBank database (http://www.ncbi.
nlm.nih.gov/blast/) to identify the bacteria.
Results
Study Population
A total of 36 cases of recurrent otitis media present in the
pediatric OPD were included in the study. The mean age of
the patients was 28.69 months (range: 11–54 months). Most
of the patients were aged 1 to 3 years (63.8%, n ¼ 23), and the
remaining were in 3 to 5 years of age group (36.1%, n ¼ 13);
88.8% patients were males (n ¼ 32) and 11.1% were females
(n ¼ 4).
All the patients had fever at presentation. On otoscopic
examination, all had tympanic membrane bulging; 85%
(n ¼ 30) had moderate to severe erythema of the tympanic
membrane; the other six cases had no tympanic membrane
erythema. About 80.5% of the children (n ¼ 29) had a history
of three episodes of otitis media in the last 6 months, and
19.4% (n ¼ 7) had four episodes. Twelve (33.3%) patients
were receiving antibiotics at the time of presentation
(►Table 1).
Bacterial Culture
Culture growth was negative for all the samples after
48 hours of incubation.
Journal of Pediatric Infectious Diseases Vol. 14 No. 3/2019
Real-Time Polymerase Chain Reaction of Clinical
Samples
The presence of pneumococci was observed in six samples.
LytA, Ply, PsaA, and spn9802 were positive in all six samples. The
cycle threshold (Ct) value and average copy number are shown
in ►Table 2.
PCRSeqTyping for Streptococcus pneumoniae
Streptococcus pneumoniae, 2 nos. belonged to serotype 19A.
cpsB PCR amplification was not observed in four qmPCR-
positive samples.
16s rDNA Sequencing
16s rDNA PCR could identify bacterial pathogens in 33 out of
36 MEF specimens. Four samples showed mixed reads on 16s
rDNA sequencing. The organisms identified were Neisseria
spp. other than N. meningitidis (n ¼ 7), N. meningitidis
(n ¼ 8), Lactococcus spp. (n ¼ 5), S. pneumoniae (n ¼ 2),
Pseudomonas aeruginosa (n ¼ 2), H. influenzae (n ¼ 1), S.
infantis (n ¼ 1), S. epidermidis (n ¼ 1), Staphylococcus aur-
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icularis (n ¼ 1), and Streptococcus sp. (n ¼ 1).
Discussion
Recurrent acute otitis media is a challenging infectious disease
in many developing countries including India. Literature on the
prevalence of the disease is limited, especially for countries
such as India. Substantial numbers of cases remain undiag-
nosed, which underestimates the incidence of recurrent otitis
media. It is generally a disease of childhood but a person of any
age can be a victim.34 It is an infection requiring antibiotic
prescription. The presence of organisms in the MEF is impor-
tant not only with regard to the etiology and pathogenesis of
recurrent otitis media but also with regard to the outcome of
the disease. The presence of organisms in the MEF may
considerably worsen both the short- and long-term outcomes
of otitis media. Untreated cases can result in a broad range of
complications such as persistent otorrhea, mastoiditis, labyr-
inthitis, meningitis, and facial nerve paralysis. Some may
develop life-threatening complications such as lateral sinus
thrombosis or brain abscess.8
Bacterial culture of MEF has been the standard for the
etiologic diagnosis of AOM. Various bacteriological studies
on rAOM have shown that the most frequently isolated
bacteria were H. influenzae, S. pneumoniae, and M. catar-
rhalis,2,35 followed by S. aureus, group A streptococci, and P.
aeruginosa in few cases.36 About 44 to 75% of MEF samples
are positive for bacterial pathogens in conventional cul-
ture.37 However, the proportions of culture-positive MEF
samples decrease in patients with rAOM37,38 and in patients
with treatment failure.37,39 Host defense mechanisms and
previous antibiotic treatments may directly contribute to
diminish survival of the pathogens in clinical samples and
diminish the likelihood of detection of bacteria by culture. In
addition, the presence of bacterial biofilms has been corre-
lated with negative bacterial cultures.37,40
Detection and identification of bacteria directly from clin-
ical samples by broad-range PCR targeting the 16S rDNA gene

followed by DNA sequencing is a valuable tool in clinical
microbiology.41 Several laboratories have used rDNA-based
molecular techniques to identify and characterize human
pathogens and commensal.42–46 These studies have identified
a plethora of microbes associated with humans, many of which
represent novel genera that previously were undescribed at
the molecular level.47
In the present study, we evaluated 16S rDNA sequencing
for diagnosis of rAOM. Neisseria spp. were found to be the
most common pathogen among the tested specimens fol-
lowed by Lactococcus spp., S. pneumoniae, H. influenzae, and
other Streptococcus spp. AOM pathogens S. pneumoniae and
H. influenzae were detected in 6 and 3% of the rAOM cases,
respectively, in the present study.
Our study detected the presence of P. aeruginosa in 6% of
the cases. The findings correlated to some extent with
studies of Butbul-Aviel et al48 and Perveen et al.49 Pseudo-
monas aeruginosa was detected as the leading pathogen of
rAOM in Israel, when the child has had recurrent episodes of
AOM in Butbul-Aviel et al’s study.48 Likewise, Perveen et al
reported that P. aeruginosa was the most common causative
microorganism of ear infection.49
Neisseria meningitidis was identified in 24% of the tested
specimens. Neisseria meningitidis has a wide range of man-
ifestations, ranging from transient mild sore throat to fatal
meningitis. Manifestations of N. meningitidis infection
include septic arthritis; upper or lower respiratory tract
infections such as otitis media, pharyngitis, bronchitis, and
pneumonia; pericarditis; myocarditis; endocarditis; and
conjunctivitis.50 Though N. meningitidis is not a commonly
isolated microbe in AOM cases, much less is known about the
true picture of recurrent otitis media pathogens among
Indian children. Neisseria meningitidis may be one of the
pathogens associated with recurrent otitis media, but cau-
tion is required in interpreting the significance of the find-
ings of our study, given the small sample size.
The gram-negative bacteria genus Neisseria includes both
pathogenic and commensal species that are found primarily in
the upper respiratory tract of humans. The genus Neisseria has
more than 20 species of gram-negative bacteria that colonize
mucosal surfaces and the oral cavity of humans. The presence of
Neisseria spp. other than N. meningitidis (21%) in the current
study indicates the possible contamination from ear microflora.
Lactococcus spp. were identified in 15% of the samples.
Although lactococci have recently been associated with
human disease,51–53 we concluded that, based on the clinical
evaluation, the lactococcal DNA was most likely a contami-
nant introduced at the time of tympanocentesis.
Staphylococcus epidermidis, S. auricularis, and Streptococ-
cus spp. are the normal bacterial flora in a healthy ear canal
and upper respiratory tract.54 These bacteria are identified in
12% of the tested samples, indicating the possible chances of
contamination of MEF specimen.
In the study, 16s rDNA sequencing is used in addition to
culture methods to identify the microbes and qmPCR to
identify S. pneumoniae in MEF samples. Culture yielded no
growth in all the samples which may be due to prior antibiotic
usage and the fastidious nature of the organisms. While the
Bacterial Profile of Middle Ear Fluid Nagaraj et al. 113
16s rDNA sequencing method detected S. pneumoniae in only
two cases, qmPCR detected S. pneumoniae in six MEF samples,
demonstrating the greater sensitivity of the qmPCR. ►Table 2
shows that qmPCR was able to detect S. pneumoniae with a
copy number as low as 10 copies/mL, while cpsB PCR failed to
amplify the target region in four samples due to low copy
number. cpsB PCR was positive in two samples and the
serotype was identified as 19A in both. The copy numbers of
four samples were 5, 16, 15, and 4 copies/mL, respectively
(►Table 2). Even though 16s rDNA PCR could amplify from
these samples, mixed reads were observed upon sequencing.
Various limitations of this study need to be considered.
First, the number of MEF samples studied was small, as it was
a pilot study to standardize the methodology and guide
future research. In addition, the cases recruited were on
antibiotic treatment limiting the usefulness of culture
method. Second, only aerobic culture method was used
and anaerobic culture was not performed. Isolation of com-
mensal organisms raises the doubt of contamination at the
time of sample collection which needs to be validated with
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repeat sample collection and identification.
The use of broad-range 16s rDNA PCR in clinical micro-
biology is a challenge. A review of the literature from Sontakke
et al55 indicates that the diagnostic utility of broad-range 16S
rDNA PCR is enhanced substantially if the detected organism is
a well-documented pathogen. Frequent detection of environ-
mental organisms of undetermined pathogenicity is currently
a limitation. The method is more vulnerable to contamination
than a species-specific PCR and no DNA extraction protocol is
available that has the same effectiveness for both gram-
positive and gram-negative bacteria. All bacterial DNA present
in a sample is amplified, including that which is unavoidably
present in reagents, meaning low-level environmental con-
tamination is impossible to eliminate entirely. At a high
number of thermal cycles, this low-level background contami-
nant DNA will be amplified and gives a false-positive result. To
reduce this risk, sequencing must be performed to distinguish
between a genuine pathogen and contaminants (often water-
borne bacteria highly unlikely to cause disease). Using stan-
dard sequencing techniques, only the most dominant DNA
sequence can be identified, which means that in samples
where more than one bacterial species are present (such as
stool), results are uninterpretable.9
The most important troubling factor in universal 16s
rDNA PCR is the contamination of samples during MEF
collection, DNA extraction, reaction mixture preparation,
amplification, and detection procedures. Using extra pure
reagents and application of RT-PCR could improve the results
of the PCR assay.56 Based on review of the literature, a more
uniform consensus on the accurate interpretation of broad-
range 16S rDNA PCR, results are needed to improve the
microbiological utility of this modality for the diagnosis of
bacterial infections in human patients.55
Conclusion
Our study demonstrates the use of broad-range 16s rDNA
sequencing method to detect microbes present in MEF. The
Journal of Pediatric Infectious Diseases Vol. 14 No. 3/2019
114 Bacterial Profile of Middle Ear Fluid Nagaraj et al.
study needs to be conducted with bigger number of samples
longitudinally to obtain relevant regional data. The study
provides insight to the etiology of bacterial pathogens in
rAOM with molecular techniques among Indian children for
the first time.
Conflict of Interest
None declared.
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