J Clin Periodontol 2013; 40: 833840 doi: 10.1111/jcpe.

12131

Oral microbial colonization in

laryngectomized patients as a
possible cofactor of biofilm
formation on their voice
prostheses

Kristina Bertl1, Beata Zatorska2,

Matthias Leonhard2, Julia Rechenmacher-Strauss2, Imme Roesner2 and
Berit Schneider-Stickler2
1

Division of Oral Surgery, Bernhard Gottlieb

School of Dentistry, Medical University of
Vienna, Vienna, Austria; 2Division of
Phoniatrics-Logopedics, Department of
Otorhinolaryngology, Medical University of
Vienna, Vienna, Austria

Bertl K, Zatorska B, Leonhard M, Rechenmacher-Strauss J, Roesner I, SchneiderStickler B. Oral microbial colonization in laryngectomized patients as a possible
cofactor of biofilm formation on their voice prostheses. J Clin Periodontol 2013;
40: 833840. doi: 10.1111/jcpe.12131.

Abstract
Aim: Biofilm formation on voice prostheses, which are used for voice rehabilitation in laryngectomized patients, is a main cause of device failure. The aim of this
study was to assess whether the presence of periodontal pathogens in the biofilm
on voice prostheses is related to that in the oral cavity and associated with the
periodontal status of the patients.
Methods: Thirty-one laryngectomized patients were invited to participate, 13 of
whom met exclusion criteria. The remaining 18 were classified according to the
community periodontal index of treatment needs (community periodontal index
of treatment needs (CPITN), grades 04). Biofilm samples from the oral
cavity and voice prostheses were analysed by PCR-based hybridization for
11 pathogens.
Results: All dentate patients required periodontal treatment (CPITN-3: n = 4,
CPITN-4: n = 8); the remaining six were edentulous. The diversity (i.e. number of
bacterial species detected) of pathogens detected on the voice prostheses correlated significantly positively with the diversity of pathogens in the oral cavity and
with clinical parameters. Furthermore, the diversity of pathogens differed
significantly between dentate and edentulous patients.
Conclusions: Results emphasize the oral cavity as an important source of bacteria
for biofilm formation on voice prostheses. Whether these pathogens reduce the
lifetime of the device by increased biofilm formation and/or increase the risk of
silicone deterioration requires further study.

Conflict of interest and source of

funding statement
All authors have no conflict of interest
and no source of funding to declare.

Laryngeal cancer is the 2nd most

common cancer of the respiratory
tract. In Europe, the incidence rate
is 10/10 000 cases in males, and the
overall 5-year survival rate is about
63% (Sokic et al. 1995, Tonetti

2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Key words: bacteria; biofilm; laryngectomized

patients; periodontitis; voice prostheses
Accepted for publication 3 June 2013

1998, Tomar & Asma 2000, Rudolph

et al. 2011). Standard therapy for
advanced-stage laryngeal and hypopharyngeal carcinoma (stages T3
T4) is still total laryngectomy, with
the main consequences being loss of

833

834

Bertl et al.

voice and impaired quality of life

(Kazi 2007, Kazi et al. 2010). Since
1980, voice rehabilitation with tracheo-esophageally inserted voice
prostheses made of medical polymers
has become an established therapy
in patients after laryngectomy
(Singer & Blom 1980). One main
complication of these voice prostheses is biofilm formation, especially
on the oesophageal valve surface.
This area cannot be cleaned by the
patient by any cleaning procedure,
and biofilm formation leads to malfunction of the prostheses, causing
aspiration (nutritional components
and saliva), and speaking difficulties.
In such cases, replacement is necessary, usually performed by a physician, approximately every 3 months
(Ackerstaff et al. 1999, Op de Coul
et al. 2000, Leonhard et al. 2010,
Reumueller et al. 2011). Hence, a
primary research focus is to improve
the lifetime of the voice prostheses,
which would significantly improve
patients quality of life. Thus far,
mainly aerobic bacteria, such as
Streptococcus spp., Staphylococcus
spp., and Candida spp. have been
investigated as integral components
of the biofilm on voice prostheses
(Ramage et al. 2006, Leonhard et al.
2009, Leonhard et al. 2010, Schuldt
et al. 2010, Reumueller et al. 2011).
In vitro models to mimic interactions
between bacteria and fungi have
been developed, but a model that
reflects the in vivo biofilm found on
voice prostheses is currently lacking.
Such models are needed to test novel
treatment strategies. Recently, our
study group identified facultative
and obligate anaerobes as part of
biofilm formation on voice prostheses (Bertl et al. 2012). These anaerobic pathogens might represent the
missing link for more accurate biofilm models. However, the diversity
(i.e. number of bacterial species
detected) of facultative and obligate
anaerobic pathogens detected was
considerable, from 10 to none, on
the voice prostheses investigated,
which raises the question of their
source. One possibility is the oral
cavity, as pathogens are well-known
components of biofilm formation in
periodontal pockets as well as on the
oral mucosa, such as the tongue
(Socransky & Haffajee 2005). Therefore, the aim of this study was to
assess the periodontal health status

of laryngectomized patients in the

re-habilitation phase after cancer
treatment, as well as their oral
microbial colonization, and to compare it with the composition of the
biofilm on their voice prostheses. We
hypothesized that a correlation exists
between
periodontal
pathogens
detected on voice prostheses and
those in the oral cavity.
Materials and Methods
Patients

The protocol for this prospective

study was approved by the ethics
committee of the Medical University
of Vienna (EK 402/2011). Patient
recruitment occurred at the outpatient department of the Department
of Otorhinolaryngology (Medical
University of Vienna) between October 2011 and June 2012. In this time
period, the 31 patients asked for participation represented the whole population of laryngectomized patients
provided with voice prostheses consulting our outpatient department
for follow-up; thus, the inclusion of
additional patients was not possible.
Antibiotic intake within the preceding 3 months, tumour recurrence,
prostheses with a lifetime of less
than 3 weeks, and voice prostheses
other than Provox2! (Atos Medical,
H
orby, Sweden) were defined as
exclusion criteria. According to these
criteria, 13 patients were excluded
(four had received antibiotics within
the preceding 3 months, four presented tumour recurrence, three
patients declined participation, and
two were provided with Blom Singer
voice prostheses). The remaining 18
patients (15 males, 3 females; mean
age ! SD, 65.7 ! 9.7 years; 12 dentate, 6 edentulous) agreed to participate and did not meet any of the
exclusion criteria. None of these
patients received periodontal treatment in the preceding 4 months.
Patients characteristics are presented
in Table 1. Only Provox 2 (Atos
Medical, H
orby, Sweden) was
included, to prevent influence due to
different surfaces or materials, and
device lifetimes were documented. In
all patients, indication for replacement was device failure due to biofilm formation, with consequent
transprosthetic leakage and aspiration.

In dentate patients (n = 12), a

single investigator (K.B.) performed
oral examination and evaluation
according to standard clinical periodontal parameters, using a periodontal probe (CP12, Hu-Friedy,
Leimen, Germany): probing pocket
depths (PD) on six sites per tooth
(disto-, mid-, and mesio-buccal and
palatinal aspects) and number of
teeth with PD >5 mm. Intra-examiner reproducibility was confirmed
by repeating PD measurements in
two randomly chosen quadrants of
five patients later during the same
appointment. The intra-class correlation coefficient yielded a value of
0.92.
The patients were further subdivided according to the community
periodontal index of treatment needs
(CPITN)
as
follows:
CPITN0 = healthy
periodontium,
no
bleeding on probing, PD <4 mm;
CPITN-1 = bleeding on probing, PD
<4 mm; CPITN-2 = presence of
supra- or subgingival dental calculus
or overhanging restorations, PD
<4 mm; CPITN-3 = PD measuring
45.5 mm; CPITN-4 = PD of 6 mm
or more; and CPITN-X = edentulism
(Ainamo et al. 1982, WHO 1997).
Oral cavity: Biofilm sampling and
determination of periodontal pathogens

From dentate patients, biofilm samples were collected subgingivally,

whereas from edentulous patients,
biofilm samples were collected from
the dorsum of the tongue, which is
described as the site with the highest
bacterial load in edentulous patients
(Sachdeo et al. 2008).
Before subgingival plaque samples were obtained from the dentate
group, supragingival plaque deposits
were removed. The sampling sites
were dried and protected from salivary contamination. Five sterile paperpoints (ISO 55) were placed at
the bottom of the five deepest periodontal pockets for 15 s. Paperpoints were stored together (pooled
sample per subject) and analysed by
means of micro-IDent! and microIDent!-plus assays in cooperation
with HAIN Lifescience (Nehren,
Germany).
In the edentulous group, samples
from the dorsum of the tongue
were collected as described by
Sachdeo et al. (2008), by means of

2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Biofilm composition on voice prostheses

835

Table 1. Patients characteristics

Patient Nr.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

Sex

Age
(years)

Diagnosis

TNM
classification*

Reconstruction
with free
jejunal flap

Radio-therapy
dose (Gy)

Insertion
of voice
prosthesis

Male
Male
Male
Male
Male
Female
Male
Male
Female
Male
Female
Male
Male
Male
Male
Male
Male
Male

76
58
72
55
71
60
70
54
55
61
73
58
56
57
84
62
83
65

Hypopharyngeal SCC
Hypopharyngeal SCC
Hypopharyngeal SCC
Laryngeal chondrosarcoma
Laryngeal SCC
Hypopharyngeal SCC
Laryngeal SCC
Hypopharyngeal SCC
Laryngeal SCC
Laryngeal SCC
Hypopharyngeal SCC
Laryngeal SCC
Hypopharyngeal SCC
Laryngeal SCC
Hypopharyngeal SCC
Hypopharyngeal SCC
Laryngeal SCC
Laryngeal SCC

T3 N2b M0
T3 N2c M0
T4 N2c M0
T1 N0 M0
T2 N0 M0
T3 N2b MX
T3 N0 M0
T4 N2c M0
T4a N2c M0
T4 N0 M0
T2 N0 M0
T3 N0 M0
T3 N2b M0
T4a N0 M0
T3 N2c M0
T4 N0 M0
T1 N0 M0
T3 N2b M0

No
Yes
Yes
No
No
Yes
Yes
No
Yes
No
No
No
No
No
No
Yes
No
No

60
42
66
None
70
60
None
66
66
60
70
None
60
60
60
60
70
60

Secondary
Secondary
Secondary
Primary
Secondary
Secondary
Secondary
Secondary
Secondary
Secondary
Secondary
Primary
Primary
Secondary
Secondary
Secondary
Secondary
Secondary

*Staged according to the AJCC/UICC Staging Manual; SCC squamous cell carcinoma.

MasterAmpTM buccal swab brushes

(Biozyme, Vienna, Austria). By gentle
stroking of the dorsum of the tongue,
microorganisms for DNA probe analysis were obtained. Samples were
stored at "40C until isolation of the
DNA with the MasterAmpTM Buccal
Swab DNA Extraction Kit (Biozyme,
Vienna, Austria). The micro-IDent!
and
micro-IDent!-plus
assays
(HAIN Lifescience GmbH, Nehren,
Germany) were used for qualitative
assessment of the presence of pathogens by a PCR-based hybridization
method. After DNA isolation, amplification by multiplex PCR with
biotinylated primers and the taq
DNA polymerase enzyme (Bioron,
Ludwigshafen, Germany) was performed. In each run, negative and
positive amplification controls were
included. Subsequently, amplified
products were chemically denatured,
and the single-stranded biotinlabelled amplicons were hybridized to
membrane-bound probes. After stringent washing, a streptavidin-alkaline
phosphatase conjugate was added
and an alkaline
phosphatasemediated staining reaction was
performed. A template ensured interpretation of the banding pattern
obtained. The detection limit was 104
for all bacteria except Aggregatibacter
actinomycetemcomitans
(detection
limit: 103). This PCR-based hybridization method has been proven to be
reliable and useful in various studies

(Eick & Pfister 2002, Haffajee et al.

2009, Eick et al. 2011).
Eleven pathogens were determined as follows:

Aggregatibacter actinomycetemcomitans
Porphyromonas gingivalis
Tannerella forsythia
Treponema denticola
Prevotella intermedia
Parvimonas micra
Fusobacterium nucleatum
Campylobacter rectus
Eubacterium nodatum
Eikenella corrodens
Capnocytophaga spp.

Voice prostheses: Biofilm sampling and

determination of periodontal pathogens

Biofilm sampling and analysis of the

voice prostheses were performed as
described previously (Bertl et al.
2012). After removal, the voice prostheses were stored in phosphatebuffered solution. Biofilm samples
were dissolved from the surface by
means of an ultrasonic cleaning system, and DNA was isolated by means
of the Maxwell! DNA Purification
Kit (Promega, Vienna, Austria).
Further processing was performed as
described above by means of microIDent! and micro-IDent!-plus assays
(HAIN Lifescience GmbH, Nehren,
Germany).

2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Statistical analysis

Possible correlations among recorded

parameters were assessed by Spearman correlation coefficient. Due to
missing normal distribution of the
data, comparison among the groups
(edentulous versus CPITN 3 versus
CPITN 4) was performed by the
KruskalWallis test. In case of significance, the MannWhitney U-test
was applied. Statistical analysis was
performed with SPSS Version 19.0
(SPSS, Chicago, IL, USA), and a
p-value <0.05 was considered statistically significant.
Results
Periodontal health status of
laryngectomized patients

Eighteen laryngectomized patients

were included in this study: 12 dentate and 6 edentulous patients. None
of the dentate patients was periodontally healthy; all 12 dentate patients
required periodontal treatment. Four
of the dentate patients presented
CPITN-3 and the remaining eight
dentate patients CPITN-4. Patients
clinical periodontal parameters are
presented in Table 2.
Biofilm composition in the oral cavity

Up to 11 different species were detectable in the subgingival biofilm

samples from the dentate patients

836

Bertl et al.

(Table 3). The frequencies of the periodontal pathogens tested are presented in Fig. 1. The most prevalent
periodontal pathogen was F. nucleatum (91.7%). Capnocytophaga spp.
(75%), P. micra (66.7%), T. forsythia,
T. denticola, and C. rectus (all 50%)
were also detected frequently.
In edentulous patients, none of
the bacteria investigated exceeded
the detection limit.
Biofilm composition on the voice
prostheses

Nine of the 11 periodontal pathogens

tested were detectable on the surfaces

of the voice prostheses examined. On

13 prostheses, up to eight periodontal
pathogens were detectable, whereas
on five prostheses no periodontal
pathogens were found. The bacterial
spectrum of each voice prosthesis is
given in Table 3.
The frequencies of the periodontal pathogens tested are presented in
Fig. 1. The most prevalent periodontal pathogen was F. nucleatum
(66.7%). Furthermore, T. forsythia,
T. denticola, P. micra, and E. corrodens were detected frequently (all
27.8%), whereas A. actinomycetemcomitans and P. intermedia could not
be found in any sample.

Associations among periodontal health

status, biofilm composition of the oral
cavity, and biofilm on the voice
prostheses

The edentulous patients had a significantly simpler microflora on their

voice prostheses (only one or two
species) in comparison with the
group
of
dentate
patients
(p = 0.003). Furthermore, on 5 of
the 18 voice prostheses, none of the
periodontal pathogens tested could
be detected; four were taken from
edentulous patients and only one
from a dentate patient (CPITN-3).
In 11 of the 18 patients, all
microbial species on the voice prostheses, as well as additional bacteria,
occurred in the oral cavity as well.
On the remaining seven voice prostheses, one or two other periodontal
pathogens could be identified on the
voice prostheses compared with the
oral cavity (Table 3).
CPITN-4 patients presented significantly higher diversity of periodontal pathogens on the voice
prostheses than did CPITN-3
(p = 0.028)
and
edentulous
(p = 0.001) patients (Fig. 2). A significant correlation could be found
between the diversity of periodontal
pathogens detected on the voice
prostheses and that in the oral cavity
(Fig. 3).

Table 2. Patients clinical periodontal parameters and indwelling time of the voice prostheses

CPITN X
(n = 6)
CPITN 3
(n = 4)
CPITN 4
(n = 8)

Indwelling
time (days)

No. of teeth*

No. of teeth
with
PD 5 mm

PD (mm)

PD of
sampling
sites (mm)

63.00 ! 64.50

138.50 ! 72.25

10.25 ! 5.68

0.50 ! 3.25

3.02 ! 0.86

4.20 ! 0.85

4.00 ! 6.00,

3.44 ! 1.51

6.10 ! 2.38

77.00 ! 108.00 14.13 ! 10.45

PD, pocket depth; CPITN, community periodontal index of treatment needs.

*Mean ! SD.

Median ! interquartile range.

Significantly higher than edentulous (p 0.01).

Significantly higher than CPITN 3 (p < 0.05).

Table 3. Occurrence of 11 periodontal pathogens on 18 voice prostheses and in the oral cavity.
CPITN

Patient No.

Indwelling time (days)

Bacterial species
A.a.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

41
24
84
98
70
308
154
29
83
123
169
154
91
185
58
64
26
62

P.g.

T.f.

T.d.

+
+

+
+

+
+

+
+

P.i.

P.m.

+
+
+
+

F.n.
+
+
+
+
+
+
+
+

C.r.

E.n.

E.c.

C.s.
+

+
+
+
+

+
+
+

+
+
+

+
+

+ Periodontal pathogen was detected on the voice prosthesis; light grey background indicates that the periodontal pathogen was detected in
the oral cavity. CPITN, community periodontal index of treatment needs. A.a, Aggregatibacter actinomycetemcomitans; P.g, Porphyromonas
gingivalis; T.f, Tannerella forsythia; T.d, Treponema denticola; P.i, Prevotella intermedia; P.m, Parvimonas micra; F.n, Fusobacterium
nucleatum; C.r, Campylobacter rectus; E.n, Eubacterium nodatum; E.c, Eikenella corrodens; and C.s, Capnocytophaga spp.
2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Biofilm composition on voice prostheses

100%
Voice prostheses
Oral cavity

Frequency

80%

60%

40%

20%

0%
A.a.

P.g.

T.f.

T.d.

P.i.

P.m.

F.n.

C.r.

E.n.

E.c.

C.s.

Bacterial species

Fig. 1. Frequency of selected periodontal pathogens on the voice prostheses and in the
oral cavity of 18 laryngectomized patients. A.a, Aggregatibacter actinomycetemcomitans; P.g, Porphyromonas gingivalis; T.f, Tannerella forsythia; T.d, Treponema denticola; P.i, Prevotella intermedia; P.m, Parvimonas micra; F.n, Fusobacterium nucleatum;
C.r, Campylobacter rectus; E.n, Eubacterium nodatum; E.c, Eikenella corrodens; and
C.s, Capnocytophaga spp.

11

Voice prostheses
Oral cavity

10

Diversity of periodontal pathogens

prostheses correlated significantly

with clinical periodontal parameters,
such as number of teeth with PD
5 mm and mean PD of sampling
sites (Table 4).

Device lifetime of voice prostheses

The mean lifetime of the voice prostheses was 96 days (xmax = 308 days;
xmin = 24 days).
No significant correlation could
be found between lifetime of the
voice prostheses and the diversity of
periodontal pathogens on the voice
prostheses or in the oral cavity
(p > 0.934). Also, the lifetimes did
not differ significantly between dentate
and
edentulous
patients
(Table 2; p > 0.293).

6
5
4
3
2
1
0
Edentulous

CPITN 3

CPITN 4

Fig. 2. Diversity of periodontal pathogens on the voice prostheses and in the

oral cavity of 18 laryngectomized
patients, separated by dental condition
(values are expressed as mean ! SD);
* significantly higher than edentulous
and CPITN 3 (p < 0.05), significantly
higher than edentulous (p < 0.01);
CPITN, community periodontal index of
treatment needs.

Furthermore, in the group of

dentate patients, the diversity of
periodontal pathogens on the voice

Discussion

The aim of this study was to assess

whether the presence of facultative
and obligate anaerobic periodontal
pathogens in the biofilm on voice
prostheses is related to that in the
oral cavity and whether it depends
on the periodontal status of laryngectomized patients. The pathogens
investigated are known as important
components of biofilm formation in
the oral cavity, and they colonize
oral surfaces, subsequently interacting to improve attachment, survival,

2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

837

and evasion of the host immune

system (Socransky & Haffajee 1991).
For example, F. nucleatum, which
plays an important role by connecting early- and late- colonizers in
oral biofilm formation (a so-called
bridge species) (Kolenbrander
et al. 2002), was detected on 67% of
voice prostheses. Due to its high
prevalence on the voice prostheses
along with other species, a comparable role was suggested for biofilm
formation on voice prostheses (Bertl
et al. 2012).
The frequency of pathogens
detected in this study was similar to
that reported in our previous study
(Bertl et al. 2012). Furthermore, the
diversity of periodontal pathogens
detected on the voice prostheses correlated significantly positively with
the diversity in the oral cavity. This
emphasizes the oral cavity as a relevant source of bacteria for biofilm
formation on voice prostheses. Oral
bacteria might easily reach the voice
prostheses by transportation in saliva, which should be regarded as an
interesting alternative medium for
pathogen assessment. In a few cases,
a higher diversity of periodontal
pathogens could be detected on the
voice prostheses compared with that
in the oral cavity; this can at least
partly be explained by the devices
prolonged lifetime, which was in five
of seven cases over 150 days in these
patients. Several factors might have
changed the oral flora in such a time
period.
Still, similar to results from our
pilot study, the diversity of pathogens detected showed great variety
among the voice prostheses investigated. This might be dependent on
the presence of teeth and periodontal
status of the laryngectomized
patients; edentulous patients harboured significantly fewer periodontal pathogens on their voice
prostheses compared with dentate
patients. In the past, it was assumed
that periodontal pathogens disappear
after all teeth have been extracted
(Danser et al. 1995, Danser et al.
1997). More recently, this opinion
has been revised: Periodontal pathogens are still detectable in the oral
cavity of edentulous patients, e.g.
from the tongue or cheek mucosa, as
well as on complete dentures (Cortelli et al. 2008, Sachdeo et al. 2008,
Fernandes et al. 2010, Kishi et al.

838

Bertl et al.

Diversity of PP on the voice prostheses

12

10

0
0

10

12

Diversity of PP in the oral cavity

Fig. 3. Correlation between the diversity of periodontal pathogens on the voice prostheses and in the oral cavity (n = 18; Spearman correlation coefficient: rho = 0.806,
p < 0.001); PP, periodontal pathogens.

Table 4. Correlations between diversity of periodontal pathogens on the voice prostheses

and clinical periodontal parameters in dentate patients (n = 12; Spearman correlation coefficient).
S

PD

rho
Diversity of
PP on the
voice prostheses

0.490

PD of
sampling sites
p-value
0.106

No. of teeth
with PD 5 mm

rho

p-value

rho

p-value

0.681

0.015

0.791

0.002

Bold values signify a significant value at the 0.05 level; PD, pocket depth; PP, periodontal
pathogens.

2010, Yasui et al. 2012). In this

study, the samples in edentulous
patients were taken from the dorsum
of the tongue, which has been
described as the oral site with the
highest bacterial load (Sachdeo et al.
2008). However, we were unable to
detect any of the 11 selected pathogens. Recent results confirmed regular
detection
of
periodontal
pathogens on the dorsum of the tongue, but not in every case. F. nucleatum was detected in every second
case, T. denticola in 40%, followed
by T. forsythia, A. actinomycetemcomitans, P. gingivalis, and P. intermedia (22% and lower) (Yasui et al.
2012). Therefore, lack of detection in
the samples of the dorsum of the
tongue in our study might be at least
partly due to the small sample size
(six edentulous patients). Still, in the
biofilm on two voice prostheses of
edentulous patients up to two different pathogens were detected. This

might be depending on the devices

lifetime, the quantity of pathogens
might steadily increase on the voice
prostheses with increasing biofilm
thickness, whereas remaining below
the detection limit on patients tongues, where the biofilm is exposed to
daily oral hygiene in contrast to the
oesophageal side of the voice prostheses, which cannot be reached by
daily cleaning procedure.
This study has an exploratory
nature with some limitations, which
should be regarded and addressed in
future trials on this topic: (i) Saliva
sampling might be preferable as it is
easy to collect (equally if patients
are dentate or edentulous) and it is
the transportation medium of oral
bacteria to the voice prostheses; (ii)
Strict case definitions of periodontitis should be applied (Page & Eke
2007); (iii) As all dentate patients
presented
periodontal
disease,
comparisons with a dentate and a

periodontally healthy control group

were not possible, which forced us to
regard the edentulous patients as the
control group. Future studies should
consider a longitudinal multicentre
study design. This would allow for
increased sample size, with a dentate
control group, and for more reliable
assessment of a possible influence on
device lifetime. Furthermore, in a
follow-up, the effect of periodontal
treatment on the biofilm composition of voice prostheses could be
addressed; (iv) The bacteria were
identified in this study by means of a
PCR-based hybridization method,
which does not allow for any differentiation between dead and vital
bacteria, or for reporting on the
location of bacteria within the biofilm. Therefore, future studies should
include other methods, such as fluorescence in situ hybridization and
confocal laser scanning microscopy
(Buijssen et al. 2012), to increase
knowledge on the position and
quantity of the bacteria within the
biofilm; And finally (v) the appearance of periodontal pathogens with
the so-far-known species of the
biofilm, such as aerobic bacteria and
Candida spp., should be addressed.
In particular, the possible interaction
of periodontal pathogens with
Candida spp. on voice prostheses is
of interest, as they are described to
interact in vitro on polymer surfaces
(Thein et al. 2006).
After diagnosis and before treatment of cancer, a patients dental situation is evaluated and, if necessary,
treated. A poor oral health status is
a risk factor for not only the development of head and neck cancer but
also for a reduced success rate of
cancer treatment and an increased
rate of side effects, such as postoperative surgical site infection
(Tezal et al. 2009, Sato et al. 2011).
The association between periodontal
disease and cancer, especially oral
and oro-pharyngeal cancer, has
recently been reviewed and underlined, although further studies following strict case definition of
periodontitis (Page & Eke 2007) are
still necessary to prove causality
(Linden & Herzberg 2013, Linden
et al. 2013). Yet, regular check-ups
in aftercare seem to be neglected, as
all dentate patients in this study
presented the necessity for repeated
periodontal treatment, with the

2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Biofilm composition on voice prostheses

majority presenting the highest
CPITN grade. This might be due to
patient non-compliance as well as to
educational or financial limitations:
for example, periodontal treatment is
not covered by national insurance in
Austria. The physicians who regularly see laryngectomized patients,
e.g. for replacing voice prostheses,
should pay attention to their
patients dental health, and regular
dental check-ups with special focus
on the periodontal situation should
be strongly recommended.
In conclusion, we confirmed our
previous results, that facultative and
obligate
anaerobic
periodontal
pathogens are regularly present in
the biofilm on voice prostheses. A
clear difference between dentate and
edentulous patients was shown;
edentulous patients harboured significantly fewer periodontal pathogens
on their voice prostheses. These
results emphasize the oral cavity,
especially the biofilm along the root
surface in deepened periodontal
pockets, as an important source of
bacteria. However, a conclusion
regarding the exact role of periodontal pathogens in biofilm formation
on voice prostheses cannot be
reached from the present results.
Whether these pathogens actually
reduce the lifetime of the device and/
or increase the risk of silicone deterioration should be addressed in
future studies.
Acknowledgements

All authors have read and approved

the manuscript, and have agreed to
its submission to this journal.
Furthermore, the authors thank Mr.
Stefan Lettner (Division of Oral
Surgery, Bernhard Gottlieb School
of Dentistry, Medical University of
Vienna, Austria) for his support in
statistical analysis.
References
Ackerstaff, A. H., Hilgers, F. J., Meeuwis, C. A.,
van der Velden, L. A., van den Hoogen, F. J.,
Marres, H. A., Vreeburg, G. C. & Manni, J. J.
(1999) Multi-institutional assessment of the
Provox 2 voice prosthesis. Archives of Otolaryngology Head and Neck Surgery 125, 167173.
Ainamo, J., Barmes, D., Beagrie, G., Cutress, T.,
Martin, J. & Sardo-Infirri, J. (1982) Development of the World Health Organization
(WHO) community periodontal index of
treatment needs (CPITN). International Dental
Journal 32, 281291.

Bertl, K., Zatorska, B., Leonhard, M., Matejka,

M. & Schneider-Stickler, B. (2012) Anaerobic
and microaerophilic pathogens in the biofilm
formation on voice prostheses: a pilot study.
Laryngoscope 122, 10351039.
Buijssen, K. J., van der Laan, B. F., van der Mei,
H. C., Atema-Smit, J., van den Huijssen, P.,
Busscher, H. J. & Harmsen, H. J. (2012) Composition and architecture of biofilms on used
voice prostheses. Head and Neck 34, 863871.
Cortelli, J. R., Aquino, D. R., Cortelli, S. C.,
Nobre Franco, G. C., Fernandes, C. B.,
Roman-Torres, C. V. & Costa, F. O. (2008)
Detection of periodontal pathogens in oral
mucous membranes of edentulous individuals.
Journal of Periodontology 79, 19621965.
Danser, M. M., van Winkelhoff, A. J., de Graaff,
J. & van der Velden, U. (1995) Putative periodontal pathogens colonizing oral mucous
membranes in denture-wearing subjects with a
past history of periodontitis. Journal of Clinical
Periodontology 22, 854859.
Danser, M. M., van Winkelhoff, A. J. & van der
Velden, U. (1997) Periodontal bacteria colonizing oral mucous membranes in edentulous
patients wearing dental implants. Journal of
Periodontology 68, 209216.
Eick, S. & Pfister, W. (2002) Comparison of
microbial cultivation and a commercial PCR
based method for detection of periodontopathogenic species in subgingival plaque samples. Journal of Clinical Periodontology 29,
638644.
Eick, S., Straube, A., Guentsch, A., Pfister, W. &
Jentsch, H. (2011) Comparison of real-time
polymerase chain reaction and DNA-strip technology in microbiological evaluation of periodontitis treatment. Diagnostic Microbiology
and Infectious Disease 69, 1220.
Fernandes, C. B., Aquino, D. R., Franco, G. C.,
Cortelli, S. C., Costa, F. O. & Cortelli, J. R.
(2010) Do elderly edentulous patients with a
history of periodontitis harbor periodontal
pathogens? Clin Oral Implants Research 21,
618623.
Haffajee, A. D., Yaskell, T., Torresyap, G., Teles,
R. & Socransky, S. S. (2009) Comparison
between polymerase chain reaction-based and
checkerboard DNA hybridization techniques
for microbial assessment of subgingival plaque
samples. Journal of Clinical Periodontology 36,
642649.
Kazi, R. (2007) Surgical voice restoration following total laryngectomy. Journal of Cancer
Research and Therapeutics 3, 188189.
Kazi, R., Sayed, S. I. & Dwivedi, R. C. (2010)
Post laryngectomy speech and voice rehabilitation: past, present and future. ANZ Journal of
Surgery 80, 770771.
Kishi, M., Ohara-Nemoto, Y., Takahashi, M.,
Kishi, K., Kimura, S. & Yonemitsu, M. (2010)
Relationship between oral status and prevalence of periodontopathic bacteria on the tongues of elderly individuals. Journal of Medical
Microbiology 59, 13541359.
Kolenbrander, P. E., Andersen, R. N., Blehert,
D. S., Egland, P. G., Foster, J. S. & Palmer,
R. J. (2002) Communication among oral bacteria. Microbiology and Molecular Biology
Reviews 66, 486505.
Leonhard, M., Moser, D., Reumueller, A., Mancusi, G., Bigenzahn, W. & Schneider-Stickler,
B. (2010) Comparison of biofilm formation on
new Phonax and Provox 2 voice prostheses a
pilot study. Head and Neck 32, 886895.
Leonhard, M., Reumuller, A., Moser, D.,
Bigenzahn, W. & Schneider-Stickler, B. (2009)

2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

839

Examination of biofilm related material deterioration on 20 PROVOX2 voice prostheses by

scanning electron microscopy. Laryngo-RhinoOtologie 88, 392397.
Linden, G. J. & Herzberg, M. C. (2013) Periodontitis and systemic diseases: a record of discussions of working group 4 of the Joint EFP/
AAP Workshop on Periodontitis and Systemic
Diseases. Journal of Clinical Periodontology 40
(Suppl), S20S23.
Linden, G. J., Lyons, A. & Scannapieco, F. A.
(2013) Periodontal systemic associations: review
of the evidence. Journal of Clinical Periodontology 40 (Suppl), S8S19.
Op de Coul, B. M., Hilgers, F. J., Balm, A. J.,
Tan, I. B., van den Hoogen, F. J. & van Tinteren, H. (2000) A decade of postlaryngectomy
vocal rehabilitation in 318 patients: a single
Institutions experience with consistent application of provox indwelling voice prostheses.
Archives of Otolaryngology Head and Neck
Surgery 126, 13201328.
Page, R. C. & Eke, P. I. (2007) Case definitions
for use in population-based surveillance of periodontitis. Journal of Clinical Periodontology 78,
13871399.
Ramage, G., Martinez, J. P. & Lopez-Ribot, J. L.
(2006) Candida biofilms on implanted biomaterials: a clinically significant problem. FEMS
Yeast Research 6, 979986.
Reumueller, A., Leonhard, M., Mancusi, G.,
Gaechter, J. N., Bigenzahn, W. & SchneiderStickler, B. (2011) Pharyngolaryngectomy with
free jejunal autograft reconstruction and tracheoesophageal voice restoration: indications
for replacements, microbial colonization, and
indwelling times of the Provox 2 voice prostheses. Head and Neck 33, 11441153.
Rudolph, E., Dyckhoff, G., Becher, H., Dietz, A.
& Ramroth, H. (2011) Effects of tumour stage,
comorbidity and therapy on survival of laryngeal cancer patients: a systematic review and a
meta-analysis. European Archives of Oto-RhinoLaryngology 268, 165179.
Sachdeo, A., Haffajee, A. D. & Socransky, S. S.
(2008) Biofilms in the edentulous oral cavity.
Journal of Prosthodontics 17, 348356.
Sato, J., Goto, J., Harahashi, A., Murata, T.,
Hata, H., Yamazaki, Y., Satoh, A., Notani, K.
& Kitagawa, Y. (2011) Oral health care reduces
the risk of postoperative surgical site infection
in inpatients with oral squamous cell carcinoma. Supportive Care in Cancer 19, 409416.
Schuldt, T., Dommerich, S., Pau, H. W. &
Kramp, B. (2010) Time course of microbial
colonization of different voice prostheses.
Laryngo-Rhino-Otologie 89, 606611.
Singer, M. I. & Blom, E. D. (1980) An endoscopic technique for restoration of voice after
laryngectomy. The Annals Otology Rhinology
Laryngology 89, 529533.
Socransky, S. S. & Haffajee, A. D. (1991) Microbial mechanisms in the pathogenesis of destructive periodontal diseases: a critical assessment.
Journal of Periodontal Research 26, 195212.
Socransky, S. S. & Haffajee, A. D. (2005) Periodontal microbial ecology. Periodontology 2000
38, 135187.
Sokic, S. I., Adanja, B. J., Marinkovic, J. P. &
Vlajinac, H. D. (1995) Risk factors for laryngeal cancer. European Journal of Epidemiology
11, 431433.
Tezal, M., Sullivan, M. A., Hyland, A., Marshall,
J. R., Stoler, D., Reid, M. E., Loree, T. R.,
Rigual, N. R., Merzianu, M., Hauck, L., Lillis,
C., Wactawski-Wende, J. & Scannapieco, F. A.
(2009) Chronic periodontitis and the incidence

840

Bertl et al.

of head and neck squamous cell carcinoma.

Cancer Epidemiology Biomarkers and Prevention 18, 24062412.
Thein, Z. M., Samaranayake, Y. H. & Samaranayake, L. P. (2006) Effect of oral bacteria
on growth and survival of Candida albicans
biofilms. Archives of Oral Biology 51, 672
680.
Tomar, S. L. & Asma, S. (2000) Smokingattributable periodontitis in the United States:
findings from NHANES III. National Health

Clinical Relevance

Scientific rationale for the study:

This study addressed periodontal
health status and oral microbial
colonization as cofactors influencing biofilm composition on voice
prostheses.
Principal findings: The diversity of
periodontal pathogens detected on
voice prostheses correlated signifi-

and Nutrition Examination Survey. Journal of

Periodontology 71, 743751.
Tonetti, M. S. (1998) Cigarette smoking and periodontal diseases: etiology and management of
disease. Annals of Periodontology 3, 88101.
WHO (1997) Oral Health Surveys: Basic Methods
4th edition, pp. 3638. Geneva: World Health
Organization.
Yasui, M., Ryu, M., Sakurai, K. & Ishihara, K.
(2012) Colonisation of the oral cavity by
periodontopathic bacteria in complete denture
wearers. Gerodontology 29, e494e502.

cantly positively with the diversity in

the oral cavity and with clinical
periodontal parameters. Clear differences in biofilm composition on
voice prostheses were detected
between dentate and edentulous
patients, and all dentate patients
required
periodontal
treatment
according to community periodontal
index of treatment needs (CPITN).

Address:
Berit Schneider-Stickler
Division of Phoniatrics-Logopedics
Department of Otorhinolaryngology
Medical University of Vienna
Waehringer Guertel 18-20, A-1090 Vienna
Austria
E-mail: berit.schneiderstickler@meduniwien.ac.at

Practical implications: Whether

periodontal pathogens influence the
lifetime of the prostheses requires
further study. All dentate patients
required periodontal treatment
(according to CPITN), which
strongly
recommends
regular
dental check-ups among laryngectomized patients.

2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd