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Contents lists available at SciVerse ScienceDirect

International Journal of Pediatric Otorhinolaryngology

journal homepage: www.elsevier.com/locate/ijporl

Recurrences in chronic tonsillitis substained by tonsillar biolm-producing

bacteria in children. Relationship with the grade of tonsillar hyperplasy
Sara Torretta a,*, Lorenzo Drago b,d, Paola Marchisio c, Maurizio Cappadona a, Vittorio Rinaldi a,
Erica Nazzari c, Lorenzo Pignataro a
a

Department of Clinical Sciences and Community Health, Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico, Universita` degli Studi di Milano, Milan, Italy
Clinical Chemistry and Microbiology Laboratory, IRCCS Galeazzi Institute, Milan, Italy
Department of Pathophysiology and Transplantation, Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico, Universita` degli Studi di Milano, Milan, Italy
d
LITA Clinical Microbiology Laboratory, L Sacco, Department of Biomedical Sciences for Health, Universita` degli Studi di Milano, Milan, Italy
b
c

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 10 August 2012
Received in revised form 19 October 2012
Accepted 22 October 2012
Available online xxx

Objectives: It has been suggested that bacterial biolms are involved in chronic tonsillar disease, but
there is a lack of strong evidence concerning their etiopathogenic role in childhood chronic tonsillar
infections. The aim of this study was to assess the presence of biolm-producing bacteria (BPB) in
tonsillar bioptic specimens taken from children with recurrent exacerbations of chronic hyperplastic
tonsillitis, and to evaluate the possible relationship between them and the patients demographic and
clinical characteristics.
Methods: 22 children (68.2% males; median age 6.5 years, range 313) with recurrent exacerbations of
chronic hyperplastic tonsillitis were included. The presence of tonsillar BPB was assessed by means of
the spectrophotometric analysis of tonsillar bioptic specimens taken during tonsillectomy between
episodes of tonsillar infection.
Results: BPB were found in 50.0% of the 44 tonsillar specimens, and Staphylococcus aureus was the most
frequent pathogen (81.8%). There was a signicant relationship (p = 0.02) between the grade of tonsillar
hyperplasy (GTH) and the presence of tonsillar BPB, with an increased relative risk (RR = 4.27, standard
error = 2.57, p < 0.01) of tonsillar BPB development in children with GTH scores of >2.
Conclusions: The ndings of this study: (1) conrm the presence of tonsillar BPB in children with
recurrent exacerbations of chronic tonsillar infections; (2) suggest that GTH is an important indicator of
the presence of tonsillar BPB; and (3) raise the question as to whether tonsillar biolm is a causative
factor or just a consequence of recurrent exacerbations of chronic hyperplastic tonsillitis.
2012 Elsevier Ireland Ltd. All rights reserved.

Keywords:
Biolm
Tonsillitis
Tonsillar hyperplasy
Children

1. Introduction
It is known that bacterial biolms are involved in the chronicity of
infections and resistance to antibiotic treatments, and therefore
have a considerable negative impact on the patients quality of life
and a signicant effect on public health [1,2]. Bacterial biolms may
play a role in various recurrent/chronic upper respiratory tract
infections, including acute and chronic middle ear diseases [3,4],
chronic adenoiditis [5], and rhinosinusitis [6], and a few studies of
limited case-series of patients have recently suggested that they are

* Corresponding author at: Department of Clinical Sciences and Community

Health, Universita` degli Studi di Milano, Fondazione IRCCS Ca` Granda Ospedale
Maggiore Policlinico, Via F. Sforza 35, 20122 Milano, Italy. Tel.: +39 0250320245;
fax: +39 0250320248.
E-mail address: sara.torretta@policlinico.mi.it (S. Torretta).

also involved in chronic tonsillar disease [79]. They have been

found in the tonsillar tissue of children with chronic infections [79]
and children with chronic tonsillar hyperplasia [7], but are more
prevalent in the former. In 2009, Stoodley et al. [10] suggested that
the tonsilloliths often associated with tonsillar inammation [11]
were actually polymicrobial biolms that are structurally heterogeneous at confocal microscopy.
However, there are few published studies of biolm formation
on tonsillar surfaces, and a lack of any strong evidence concerning
the etiopathogenic role of tonsillar biolm in childhood chronic
tonsillar infections. The aim of this study was to assess the
presence of biolm-producing bacteria (BPB) in tonsillar bioptic
specimens taken from children with recurrent exacerbations of
chronic hyperplastic tonsillitis during tonsillectomy between
episodes of tonsillar infections, and evaluate the possible
relationship between their presence and the patients demographic and clinical characteristics.

0165-5876/$ see front matter 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijporl.2012.10.018

Please cite this article in press as: S. Torretta, et al., Recurrences in chronic tonsillitis substained by tonsillar biolm-producing bacteria
in children. Relationship with the grade of tonsillar hyperplasy, Int. J. Pediatr. Otorhinolaryngol. (2012), http://dx.doi.org/10.1016/
j.ijporl.2012.10.018

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2. Methods

2.6. Biolm evaluation

2.1. Study design

The biolms were evaluated using the spectrophotometric

methods of Christensen et al. [16]. In order to ensure biolm
development, after 18 h of incubation, 20 mL of the bacterial
cultures was added to a well in a 96-well polystyrene tissue culture
plate (Falcon, San Jose, California) containing 180 mL of an
appropriate growth medium (tryptic soy broth or haemophilus
test medium), and incubated for a further 18 h at 37 8C in ambient
air or a 10% CO2-enriched atmosphere. After the biolms had
developed, the growth medium was discarded, and each well was
washed three times with 200 mL of sterile normal saline to
eliminate any unbound bacteria. The adherent organisms were
then stained with crystal violet for 15 min, after which any excess
stain was rinsed off using 200 mL of distilled water.
When the plates had dried, the optical densities (ODs) of the
stained adherent bacterial biolms were read by a single trained
examiner (LD) using a microplate reader (model 680, BIORAD,
Segrate, Milan, Italy) at 595 nm. Adherence was measured in
triplicate, and the values were averaged. On the basis of the ODs
and the cut-off OD (ODc; dened as three standard deviations
above the mean OD of the negative control), the BPB strains were
classied into four categories on the basis of their bacteria-biolm
producing capacity: biolm non-producers (OD  ODc), weak
biolm producers (ODc < OD  2  ODc), moderate biolm producers (2  ODc < OD  4  ODc), and strong biolm producers
(4  ODc < OD) [17]. The samples were considered positive for BPB
when at least one specimen per patient was positive.

A longitudinal study at a tertiary hospital.

2.2. Study setting
The study was carried out at the Department of Clinical Sciences
and Community Health and at the Department of Pathophysiology
and Transplantation of Fondazione IRCCS Ca Granda Ospedale
Maggiore Policlinico (Milan, Italy). The protocol was approved by
our local Ethics Committee, and written informed consent was
obtained from the childrens parents or legal guardians.
2.3. Study subjects
Between March 2008 and February 2009, we recruited
outpatients aged 313 years who were referred to our Division
of Otolaryngology for tonsillectomy because of recurrent
exacerbations of chronic tonsillitis dened as severe recurrent
tonsillar infections (at least seven episodes of acute tonsillitis in
the preceding year, at least ve episodes per year for two
consecutive years, or at least three episodes per year for three
consecutive years or more) [12] with chronic tonsillar hyperplasy dened by a grade of tonsillar hyperplasy (GTH)  1
according to Brodsky [13].
The exclusion criteria were previous adenoidectomy; acute
febrile illness; upper respiratory tract infection or antibiotic
therapy in the previous 14 days; concomitant systemic diseases;
and craniofacial, neuromuscular, immunological, syndromic or
dened genetic abnormalities. Fourteen days were chosen because
they are a longer period compared with 10 days, which are the
current standard duration of antibiotic treatment in case of acute
tonsillitis [14], and on the basis of our previous studies on
paediatric otolaryngological chronic infections related to biolm
[5,15].

2.7. Statistical analysis

The prevalence and distribution of the BPB were assessed. The
relationship between the presence of BPB in the tonsillar specimen
and the demographic and clinical variables was tested by means of
Fishers exact probability test and logistic regression analysis.
The data were analyzed using STATA 10.0 software (StataCorp,
College Station, TX, USA).

2.4. Intervention and specimen collection

3. Results

A record was made of each patients demographic and clinical

characteristics, including the number of the documented tonsillar
infections in the previous 12 months, and the GTH according to
Brodsky [13]. The GTH was scored by the same trained examiner
(ST) as follows: grade 0 = tonsils in the tonsillar fossa, with no
impingement on the oropharyngeal airway; grade 1 = tonsils just
outside the tonsillar fossa, obstructing less than 25% of the airway;
grade 2 = tonsils readily seen and obstructing 2550% of the
airway; grade 3 = tonsils obstructing 5075% of the airway; and
grade 4 = tonsils obstructing >75% of the airway.
Bilateral tonsillar biopsies were performed during tonsillectomy under general anaesthesia for BPB searches. In the case of acute
infection, tonsillectomy was postponed until its complete clinical
resolution (at least 14 days after remission).

BPB were detected in 50.0% of the 44 tonsillar specimens taken

from the 22 enrolled children (68.2% males; median age 6.5 years,
range 313 months). The main pathogen was Staphylococcus
aureus, which was detected in 18 samples (81.8%). Table 1 shows
the main demographic and clinical data.
Table 2 shows the microbiological results by GTH. The presence
of tonsillar BPB did not correlated with gender, age, or the number
of reported tonsillar infections in the previous 12 months, but
there was a signicant relationship (p = 0.02) with GTH, with an
increased relative risk of tonsillar BPB development (RR = 4.27,
standard error = 2.57, p < 0.01) in the children with GTH scores of
>2. Being a child with a GTH of >2 was conrmed to be the only
predictor of the presence of tonsillar BPB (odds ratio = 10.0,
standard error = 8.55) at logistic regression analysis (p < 0.01,
R2 = 15.6%).
There was no GTH-related difference in the distribution of the
BPB strains or their biolm-producing capacity, regardless of
whether the GTH was considered as a four-level variable or
dichotomous variable (Table 2). In addition, there was no
relationship between the GTH and the number of reported
tonsillar infections in the previous 12 months.

2.5. Bacterial growth and identication

All of the biopsy samples were inoculated into Stuart transport
medium tubes (Venturi Transystem, Brescia, Italy) and processed
within 2 h by the Clinical Microbiology Laboratory. They were
diluted in 1.6 or 0.5 mL of sterile normal saline, and 100 mL of these
solutions was plated on mannitol salt agar (MSA), chocolate agar
(CA), MacConkey agar (MC), and tryptic soy agar (TSA) with the
addition of 5% sheep blood. The TSA and CA plates were incubated
at 37 8C in a 10% CO2-enriched atmosphere for 18 h; the MSA and
MC plates were incubated at 37 8C for 18 h.

4. Discussion
Our ndings documented the presence of BPB in 50% of tonsillar
bioptic specimens taken during tonsillectomy from children with

Please cite this article in press as: S. Torretta, et al., Recurrences in chronic tonsillitis substained by tonsillar biolm-producing bacteria
in children. Relationship with the grade of tonsillar hyperplasy, Int. J. Pediatr. Otorhinolaryngol. (2012), http://dx.doi.org/10.1016/
j.ijporl.2012.10.018

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Table 1
Demographic and clinical features of the study group.
No. (%)
No. of children
Males
Median age, years (range)
Median number of tonsillar infectionsa (range)
GTH
0
1
2
3
4
BPB in tonsillar specimen
Bacterial strain
S. aureus
S. pyogenes
H. inuenzae
P. aeruginosa
Biolm-producing capacity
Weak
Moderate
Strong

22
15 (68.2)
6.5 (313)
4 (35)
0
4
9
21
10
22

(0)
(9.1)
(20.5)
(47.7)
(22.7)
(50.0)

18
2
1
1

(81.8)
(9.1)
(4.5)
(4.5)

8 (36.4)
7 (31.8)
7 (31.8)

GTH: grade of tonsillar hypertrophy according to Brodsky [13]; BPB: biolmproducing bacteria.
a
In the previous 12 months.

recurrent exacerbations of chronic hyperplastic tonsillar infections. This conrms previous ndings [79] and seems to be in line
with the reported prevalence of tonsillar biolm (4185%) in
limited case-series of patients with recurrent or chronic tonsillar
disease [79]. Involvement of bacterial biolm in a consistent
proportion of children with recurrent exacerbations of chronic
tonsillar infections suggests the need to reconsider the concepts of
chronic and recurrent infections. As a matter of fact, in case of
documented tonsillar biolm also between acute episodes (as
occurred in 50% of our samples), one may believe that tonsillar
tissue could be chronically infected by resistant BPB which would
be not cleared between recurrences and predispose to recurrent
tonsillar inammations (related to periodic spreading of planktonic species) [1,4]. Under this condition, it would be better to
speak about chronic tonsillitis with recurrent inammatory
events.
The variability in the prevalence of tonsillar biolm reported by
Literature [79] may be at least partially due to differences in
inclusion criteria because when children with chronic tonsillar
infections are considered separately from those with chronic
tonsillar hyperplasy without any infections, the results become
more homogeneous and the prevalence of tonsillar biolms is
respectively 6085% (mean 76.5%, 39/51 samples) [79] and 41%
(18/44 samples in only one study) [9].
The prevalence of tonsillar biolm documented in our study is
lower than that reported by others [79], probably because of the

different diagnostic procedures used for detection: we assessed the

existence of BPB by means of quantitative spectrophotometry [16],
whereas the other authors directly evaluated the presence of
bacterial biolm by means of advanced microscopic analyses such
as scanning and transmission electron microscopy [79]. Although
electron microscopy may be preferable for studying biolms
because it offers detailed insights into the ultrastructure of
bacteria and the environment in which they are embedded, it is
quite expensive and makes it difcult to quantify the adherent
microorganisms [18]. Spectrophotometry is a simpler and more
cost-effective means of quantifying biolm formation by evaluating the optical density of the stained BPB adhering to the smooth
surface of plastic tissue culture plates [16].
In addition to the different microbiological analyses, variations
in tissue samples may also have contributed to the lower
prevalence of tonsillar BPB in our study. We used microbiological
methods to search for BPB in small tissue samples from tonsillar
biopsies taken during tonsillectomy. On the contrary, Chole and
Faddis [7] evaluated biolm formation on the basis of the
histomorphological appearance of whole human tonsils obtained
during tonsillectomy, whereas Galli et al. [8] and Al-Mazrou and
Al-Khattaf [9] refer to tissue samples, thus making it unclear
whether they analyzed small bioptic specimens or whole tonsils.
On the basis of previous studies of biolm in recurrent or
chronic childhood upper respiratory tract infections [6], it can be
argued that, even in biolm-positive patients, only a portion of the
tonsillar surface would be covered. This hypothesis has never been
tested on tonsillar samples as the only study clearly stating that the
presence of biolm was sought on whole tonsils [7] does not
specify how much of the surface was covered. On the other hand, it
has been found that most, but not all the adenoid surface was
covered with biolm in children with chronic rhinosinusitis [6].
In our case series, S. aureus was the most frequently detected
BPB (81.8%), followed at a considerable distance by Streptococcus
pyogenes (9.1%). This conrms the ndings of previous studies
suggesting that Gram-positive organisms are predominant in
tonsillar biolm production [79]. In particular, Al-Mazrou and AlKhattaf [9] found that Staphylococcus species were the most
frequently isolated bacteria followed by Streptococcus species,
whereas Galli et al. [8] reported the opposite. The involvement of S.
aureus in biolm-related chronic tonsillar infections here discovered could be partially explained on the basis of its possible
copathogenicity presumably predisposing to infectious recurrences, as suggested by Pichichero et al. [19].
Our analysis documented a signicant relationship between
tonsillar BPB and the GTH. Being a child with larger tonsils was the
only predictor of the presence of tonsillar BPB, as the children with
a GTH of >2 had a 4.27-fold increased risk of developing tonsillar
BPB than those with smaller tonsils. To the best of our knowledge,
this association has never been tested before in studies of tonsillar
biolms. However, the nding of an increased risk in children with

Table 2
Microbiological results by grade of tonsillar hypertrophy (GTH).
GTH

1
2
3
4

No. with BPB (%)

pa

1(25.0)
1 (11.1)
14 (66.7)
6 (60.0)

GTH classes
2
2 (15.3)
>2
20 (64.5)

0.02

<0.01

pa

Bacteria (%)
S. aureus

S. pyogenes

H. inuenzae

P. aeruginosa

1
1
11
5

0
0
2
0

0
0
1
0

0
0
0
1

(100.0)
(100.0)
(78.6)
(83.3)

2 (100.0)
16 (80.0)

(0.0)
(0.0)
(14.3)
(0.0)

0 (0.0)
2 (10.0)

(0.0)
(0.0)
(7.1)
(0.0)

0 (0.0)
1 (5.0)

(0.0)
(0.0)
(0.0)
(16.7)

0 (0.0)
1 (5.0)

Biolm-producing capacity (%)

Weak

Moderate

Strong

n.s.

0
0
6
2

0
0
4
3

1
1
4
1

n.s.

0 (0.0)
8 (40.0)

(0.0)
(0.0)
(42.8)
(33.3)

(0.0)
(0.0)
(28.6)
(50.0)

0 (0.0)
7 (35.0)

pa

(100.0)
(100.0)
(28.6)
(16.7)

n.s.

2 (100.0)
5 (25.0)

n.s.

n.s.: not signicant.

a
Fishers exact probability test.

Please cite this article in press as: S. Torretta, et al., Recurrences in chronic tonsillitis substained by tonsillar biolm-producing bacteria
in children. Relationship with the grade of tonsillar hyperplasy, Int. J. Pediatr. Otorhinolaryngol. (2012), http://dx.doi.org/10.1016/
j.ijporl.2012.10.018

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larger tonsils agrees with the report of Webb et al. [20], who
showed that tonsillar size was an important indicator of recurrent
acute tonsillitis as the children with a history of tonsillitis
generally had larger tonsils (a mean GTH = 2.4; 95% CI 2.22.7)
than those without (mean GTH = 1.0; 95% CI = 0.81.2), with a cutoff point of two as in our sample.
All of our patients underwent tonsillectomy because recurrent
exacerbation of chronic tonsillar infections, and so the number of
acute events may have accounted for the difference. However, we
did not nd any signicant association between the reported
number of inammatory events in the previous 12 months and the
GTH or the presence of tonsillar BPB. This is in line with a previous
work by Weir [21], who did not nd any correlation between
tonsillar size and the number of sore throat attacks in the
preceding 12 months.
Our nding that children with larger tonsils are at increased risk
of developing biolm may have various explanations. First of all, it
can be assumed that the probability of biolm is higher in patients
with large tonsils and chronic tonsillar infections on the basis of
the fact that an opportunistic tonsillar biolm has also been
found in children with obstructive hyperplastic tonsils without any
previous infections [9].
Moreover, it is well known that bacterial adhesion to biotic
surfaces (the rst step in biolm development) is enhanced when
the mucosa is impaired [22]. This is explained in the etiopathogenic model for the biolm produced by Pseudomonas aeruginosa in
the lower airways of patients with cystic brosis, in whom
epithelial inammation and the damage caused by previous
Haemophilus inuenzae colonization pave the way for chronic
infection and biolm production by P. aeruginosa [22]. It can be
argued that the larger the tonsillar surface (on which chronic
infections due to planktonic pathogens induce recurrent inammation and activate host defense responses that lead to mucosal
damage), the greater the probability of bacterial adhesion and
consequent biolm development. In this case, the tonsillar biolm
would not act as the primum movens of chronic tonsillar infection,
but be a consequence of it.
Finally, we can assume that in patients with documented
tonsillar BPB, chronic tonsillar colonization by BPB (mainly cocci) is
responsible for the periodic release of planktonic strains that
induce acute exacerbations in patients with chronic hyperplastic
tonsillitis. Each recurrence would evoke the activation of innate
and adaptive immune factors that mediate inammation. However, neither usual antibiotic treatments nor activation of the host
defense system can overcome BPB within their complex threedimensional polymeric extra-cellular matrix because of the sporelike state of bacterial metabolic dormancy, the low replication rate,
and the acquisition of phenotypical modications inducing
resistance [23,24]. This leads to repeated inammatory mucosal
damage that enhances bacterial adhesion and subsequent biolm
production. Moreover, recurrent infections may induce some
particular histomorphological changes (including vasodilatation,
capillary leakage, mediator extravasation and brous tissue
deposition) resulting in acute tonsil swelling [20]. Repeated
tonsillar infections sustained by bacterial biolm would therefore
not only give rise to possible treatment failures [25], but also to
chronic tonsil enlargement even in the absence of acute
inammation.
A possible limitation of this study is the use of simple and costeffective quantitative spectrophotometric analysis for BPB research [16], compared to more used, expensive and complex
electron microscopic assessment [18] which could partially
inuence our results, thus suggesting caution in their direct
application in clinical practice.
In conclusion, the ndings of this study: (1) conrm the
presence of tonsillar BPB in children with recurrent exacerbation of

chronic hyperplastic tonsillitis; (2) suggest that GTH is an

important indicator of the presence of tonsillar BPB; and (3) raises
the question as to whether tonsillar biolm is a causative factor or
just a consequence of recurrent exacerbation of chronic hyperplastic tonsillitis, which can only be answered by further studies of
larger case series.
Finally, the presence of tonsillar bacteria embedded in biolm
during the interval between episodes of tonsillar infection raises
some doubts about the adequacy of the concept of acute and
chronic tonsillar infection and, if our preliminary data will be
conrmed in larger series of patients, it may be necessary to
reconsider the classication of acute, recurrent and chronic disease
on the basis of the presence or absence of tonsillar BPB, regardless
of the number of acute episodes.
Financial disclosure
The authors have no sponsorships or competing interests to
disclose in relation to this article.
Conict of interest
None.
References
[1] P.S. Stewart, J.W. Costerton, Antibiotic resistance of bacteria in biolms, Lancet
358 (2001) 135138.
[2] R.M. Donlan, Biolms: microbial life on surfaces, Emerg. Infect. Dis. 8 (2002) 881
890.
[3] J.C. Post, P. Stoodley, L. Hall-Stoodley, G.D. Ehrlich, The role of biolms in
otolaryngologic infections, Curr. Opin. Otolaryngol. Head Neck Surg. 12 (2004)
185190.
[4] Y.C.C. Liu, J.C. Post, Biolms in pediatric respiratory and related infections, Curr.
Allergy Asthma Rep. 9 (2009) 449455.
[5] S. Torretta, L. Drago, P. Marchisio, R. Mattina, I.A. Clemente, L. Pignataro, Diagnostic accuracy of nasopharyngeal swabs in detecting biolm-producing bacteria
in chronic adenoiditis: a preliminary study, Otolaryngol. Head Neck Surg. 144
(2011) 784788.
[6] J. Coticchia, G. Zuliani, C. Coleman, M. Carron, J. Gurrola 2nd, M. Haupert, et al.,
Biolm surface area in the pediatric nasopharynx: chronic rhinosinusitis vs
obstructive sleep apnea, Arch. Otolaryngol. Head Neck Surg. 133 (2007) 110114.
[7] R.A. Chole, B.T. Faddis, Anatomical evidence of microbial biolms in tonsillar
tissues: a possible mechanism to explain chronicity, Arch. Otolaryngol. Head Neck
Surg. 129 (2003) 634636.
[8] J. Galli, L. Calo`, F. Ardito, M. Imperiali, E. Bassotti, G. Fadda, et al., Biolm formation
by Haemophilus inuenzae isolated from adeno-tonsil tissue samples, and its role
in recurrent adenotonsillitis, Acta Otorhinolaryngol. Ital. 27 (2007) 134138.
[9] K.A. Al-Mazrou, A.S. Al-Khattaf, Adherent biolms in adenotonsillar diseases in
children, Arch. Otolaryngol. Head Neck Surg. 134 (2008) 2023.
[10] P. Stoodley, D. Debeer, M. Longwell, L. Nistico, L. Hall-Stoodley, B. Wenig, et al.,
Tonsillolith: not just a stone but a living biolm, Otolaryngol. Head Neck Surg. 141
(2009) 316321.
[11] B.W. Neville, D.D. Damm, C.M. Allen, J.E. Bouquot, Oral and Maxillofacial
Pathology, WB Saunders, Philadelphia, USA, 2002.
[12] J.L. Paradise, C.D. Bluestone, R.Z. Bachman, D.K. Colborn, B.S. Bernard, F.H. Taylor,
et al., Efcacy of tonsillectomy for recurrent throat infection in severely affected
children: results of parallel randomized and nonrandomized clinical trials, N.
Engl. J. Med. 310 (1984) 674683.
[13] L. Brodsky, Modern assessment of tonsils and adenoids, Pediatr. Clin. North Am.
36 (1989) 15511569.
[14] E. Chiappini, N. Principi, N. Mansi, A. Serra, S. De Masi, A. Camaioni, et al., Italian
Panel on the Management of Pharyngitis in Children. Management of acute
pharyngitis in children: summary of the Italian National Institute of Health
guidelines, Clin. Ther. 34 (2012) 14421458.
[15] L. Drago, E. De Vecchi, S. Torretta, R. Mattina, P. Marchisio, L. Pignataro, Biolm
formation by bacteria isolated from upper respiratory tract before and after
adenotonsillectomy, APMIS 120 (2012) 410416.
[16] G.D. Christensen, W.A. Simpson, J.J. Younger, Adherence of coagulase-negative
staphylococci to plastic tissue culture plates: a quantitative model for the
adherence of staphylococci to medical devices, J. Clin. Microbiol. 22 (1985)
9961006.
[17] H. Faden, M.J. Waz, J.M. Bernstein, L. Brodsky, J. Stanievich, P.L. Ogra, Nasopharyngeal ora in the rst three years of life in normal and otitis-prone children,
Ann. Otol. Rhinol. Laryngol. 100 (1991) 612615.
[18] C. Hanning, M. Follo, E. Hellwig, A. Al-Ahmad, Visualization of adherent microorganisms using different techniques, J. Med. Microbiol. 59 (2010) 17.

Please cite this article in press as: S. Torretta, et al., Recurrences in chronic tonsillitis substained by tonsillar biolm-producing bacteria
in children. Relationship with the grade of tonsillar hyperplasy, Int. J. Pediatr. Otorhinolaryngol. (2012), http://dx.doi.org/10.1016/
j.ijporl.2012.10.018

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[19] M.E. Pichichero, J.R. Casey, T. Mayes, A.B. Francis, S.M. Marsocci, A.M. Murphy,
et al., Penicillin failure in streptococcal tonsillopharyngitis: causes and remedies,
Pediatr. Infect. Dis. J. 19 (2000) 917923.
[20] C.J. Webb, E. Osman, S.K. Ghosh, S. Hone, Tonsillar size is an important indicator of
recurrent acute tonsillitis, Clin. Otolaryngol. Allied Sci. 29 (2004) 369371.
[21] N.F. Weir, Clinical interpretation of tonsillar size, J. Laryngol. Otol. 86 (1972)
11371144.
[22] T.D. Starner, N. Zhang, G. Kim, M.A. Apicella, P.B. McCray Jr., Haemophilus
inuenzae forms biolms on airway epithelia: implications in cystic brosis,
Am. J. Respir. Crit. Care Med. 174 (2006) 213220.

[23] J.W. Costerton, B. Ellis, K. Lam, F. Johnson, A.E. Khoury, Mechanism of electrical
enhancement of efcacy of antibiotics in killing biolm bacteria, Antimicrob.
Agents Chemother. 38 (1994) 28032809.
[24] G. Borriello, E. Werner, F. Roe, A.M. Kim, G.D. Ehrlich, P.S. Stewart, Oxygen
limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biolms, Antimicrob. Agents Chemother. 48 (2004) 26592664.
[25] J. Conley, M.E. Olson, L.S. Cook, H. Ceri, V. Pahn, H.D. Davies, Biolm formation by
group A streptococci: is there a relationship with treatment failure? J. Clin.
Microbiol. 41 (2003) 40434048.

Please cite this article in press as: S. Torretta, et al., Recurrences in chronic tonsillitis substained by tonsillar biolm-producing bacteria
in children. Relationship with the grade of tonsillar hyperplasy, Int. J. Pediatr. Otorhinolaryngol. (2012), http://dx.doi.org/10.1016/
j.ijporl.2012.10.018