Gerodontology. 2016 Sep;33(3):322-7. doi: 10.1111/ger.12156. Epub 2014 Nov 12.

Original Article

Potential respiratory pathogens

colonisation of the denture plaque of
patients with chronic obstructive
pulmonary disease
Authors
 Dorota Przybyłowska,

 Elżbieta Mierzwińska-Nastalska,

 Ewa Swoboda-Kopeć,
 Renata Rubinsztajn,
 Ryszarda Chazan

Abstract
Introduction

The role of bacterial infections in acute exacerbations of chronic obstructive pulmonary

disease (COPD) is widely examined. Denture plaque in patients with COPD is an example
of bacterial and fungal biofilm, which is a reservoir of potentially pathogenic respiratory
tract microorganisms. Poor denture hygiene might cause acute exacerbations of COPD.

Objective
Assessment of prevalence of respiratory tract pathogens in denture plaque in stable patients
with COPD and it influence on oral ontocenoses depending upon the therapy.

Materials and methods

The study was based on the clinical assessment of oral mucosa and denture hygiene in 53
patients with COPD with mean age of 70 ± 18 years and 14 generally healthy participants
with mean age of 65 ± 14 years. Microbiological and mycological tests were performed by
culturing direct denture swabs.

Results

The study showcased the presence of potential pathogenic micro-organisms in denture

plaque of 48 patients with COPD (90%) and nine healthy subjects (64.3%). Yeast-like fungi
prevailed in denture surface swabs of 40 (75%) in patients with COPD and 8 (57%) in cases
of control group. In 66% of patients, various degree of oral mucosa inflammation prevailed.

Conclusions

Denture plaque could be a potential source of bacterial and fungal infections in patients
with COPD.

Introduction
Chronic obstructive pulmonary disease (COPD) is one of many diseases where oral bacteria
play an important part in exacerbations. International studies showed poor oral hygiene
played a part in pneumonia infections. A substantial part of nosocomial pneumonias starts
with the aspiration of oral bacteria from the mouth or nasopharynx to the lower respiratory
tract[1-5]. Respiratory tract pathogens such as Streptococcus pneumoniae, Haemophilus
influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Escherichia coli, Proteus
mirabilis and others, and also Candida spp. fungi, leading a saprophytic existence in the
mucosa[6] are responsible for 55% of exacerbations of COPD. Those micro-organisms
exist not as single cells but as organised structures forming an ecological niche called
biofilm. The pathogenicity of a microorganism is reflected in its capacity to form biofilm,
which directly affects oral tissues and the lower respiratory tract. Microorganisms might
cause antigen release including endotoxins, lipoproteins, peptidoglycans and other
molecules, which increases inflammatory reactions in the respiratory tract and elsewhere.

Candida albicans is a good example as it secretes tyrosol, an autoinducer, stimulating

hyphal growth during intermediate growth stages and protects fungal cells against a
decrease in the expression of genes required for DNA replication. Farnesol, which inhibits
hypha formation and protects the microorganism against hydrogen peroxide, has different
properties[7].

The impairment of humoral and cellular response, resulting from chronic antibioticotherapy
and immunosuppressive drug use, affects not only patients with chronic diseases, including
renal insufficiency, diabetes, neoplasms, COPD, but also patients after transplantations.
Neutropenia and lymphocyte and phagocyte impairment promote infections caused by
microorganisms leading a saprophytic existence, and is usually its primary cause[8].

Patients with COPD are at high risk of bacterial and fungal infections, especially Candida
spp. A chronic topical oral inflammation and a systemic inflammation impede the
mucociliary clearance of the patient, which promotes further respiratory tract colonisation.
Denture stomatitis is one of the reasons for chronic topic inflammations of oral mucosa in
elderly patients wearing dentures. Removable dentures promote oral bacterial and fungal
growth. The extent of the denture plate obstructs saliva flow, impedes its antiseptic
properties, limits oxygen supply to mucosal epithelial cells, lowers the pH and increases the
temperature and the quantity of remaining food particles[3, 9]. Elderly patients use their
dentures over long periods of time, without reporting for checkups to have their dentures
lined or exchanged. Therefore, the resulting poor denture stability and retention are
traumatogenic factors for the oral mucosa. The damaged epithelia under the denture base
might become the starting point for bacterial or fungal infections.

Elderly people, using acrylic removable dentures, prevail among patients with COPD. This
biodegradable material is porous and rough, which promotes microorganism colonisation.
The mycelial form of Candida displays a strong adherence to acrylic surfaces, by means of
phospholipases and other hydrolytic enzymes present in the pseudohyphae. Poor oral and
denture hygiene promote bacterial deposits. Denture biofilm is a mix of fungal and bacterial
biofilm where complex interactions occur between C. albicans and other oral
microorganisms[3, 10].

Long-term inhaled glucocorticoid therapy and the frequent necessity of including systemic
glucocorticoids and antibiotics in case of disease exacerbation influence the immune
system[11]. Chemotherapeutics cause mouth dryness, which means denture and tooth
surfaces self-cleaning properties become significantly limited.

Objective
The objective of this study was to assess the prevalence of selected pathogens of the
respiratory tract in denture plaque, cultured from removable denture from stable patients
with COPD, undergoing various treatments. It also meant to determine the denture plaque
biofilm influence on oral ontocenoses patients depends on COPD treatment.

Materials and methods

The study was conducted on 53 (20 women and 33 men) patients with COPD (diagnosed
according to GOLD 2011 guidelines), treated at the Department of Internal Medicine,
Pulmonology and Allergology, Warsaw Medical University. Exclusion criteria were
infections in the last 3 months and antibioticotherapy. Fourteen generally healthy, not
undergoing any treatments, and regularly reporting for checkups participants (10 women
and four men) from the Department of Prosthodontics, Warsaw Medical University, were
part of the control group. The mean age among COPD participants was 70 years (53–84
years) and 65 years (51–84 years) in the control group. All participants used removable
dentures: complete, partial or cobalt-chromium removable partial denture. The study
population was divided into three groups of patients with COPD, which were treated with
bronchodilators (n = 18, 26.9%), inhaled glucocorticosteroids ICS (n = 21, 31.3%) and
home oxygen therapy (n = 14, 20.9%) and control group participants (n = 14, 20.9%).

All patients answered questions about their medical records and underwent a physical
examination. Earlier medical documentation was also analysed, including additional tests
(chest X-rays) and previous hospital discharge papers. The study was performed with
ethical committee approval.

Dental history questions included subjective symptoms within oral mucosa, denture
hygiene and diet. The oral mucosa was clinically assessed according to a modified Newton
classification. Type 0 included patients with no inflammatory changes in oral mucosa. Type
1 included patients with pinpoint hyperaemia. Type 2 included patients with a more diffuse
erythema involving mucosa covered by the denture. Type 3 included patients with mucosal
hyperplasia close to the denture.

Denture hygiene was determined according to Ambjörsen's modified plaque index and
assessed plaque levels in three areas of the denture surface and two of the working side
surfaces[12]. The hygiene levels were as following: 0, good hygiene – no plaque at
examination or probing; 1, insufficient hygiene – moderate plaque covering part of the
denture, visible at examination; 2, poor hygiene – denture covered in visible plaque.

Direct swabs were collected from acrylic denture surfaces and used for microbiological
tests. They were then sent in Transwab MD containers to the Department of Medical
Microbiology of Warsaw Medical University, where they were cultured and isolated on
liquid and gel medium, using Columbia agar with 5% sheep blood and Chapman and
MacConkey agar. Plates were incubated for 24–48 h at 37°C in the presence of oxygen.
Bacteriological identification was performed according to morphological and biochemical
characteristics, using the following bioMeriéux tests (bioMeriéux SA, Marcy-l'Etoile,
France): ID32 GN, API20 E, API20 Strep and API20 Staph. Sabouraud agar was used to
grow and identify yeast-like fungi, which were incubated at 30°C for 48 h in the presence
of oxygen. CHROMagar Candida selective medium (Bio-Rad, Marnes-la-Coquette,
France) and the assimilative ID32 C strip (bioMeriéux) were used for fungal growth
analysis and identification. All microbiological sampling was conducted by a single
examiner. There were no duplications of treatment.

The analysis of variance (anova) was performed to compare differences between the
analysed characteristics in different patient groups. The chi-squared test was used to verify
the correlations.

The authors declare no conflict of interests in relation to this article.

Results
Sixty-seven direct denture swabs underwent laboratory analysis. Participants used complete
(68.6%), acrylic partial (19.4%) and cobalt-chromium removable partial dentures (12%).
Figure 1 presents the prevalence of various denture types in COPD and healthy participants
using dentures. The majority respondents in both study populations wore complete
dentures; in COPD group, it was 66% and in control group 78.6% (Fig. 1).

Figure 1.

Type of prosthodontic appliance used in respective groups.

During the oral mucosa clinical assessment according to the Newton classification, various
inflammatory changes were detected in 44 (65.67%) patients. Type 1 in form of pinpoint
hyperaemia prevailed in 26 (49%) patients with COPD and in 2 (14.3%) participants from
the control group. A diffuse mucosal inflammation involving all the area covered by the
denture was observed in 12 (22.64%) of participants with COPD. A papilloma localised in
the central part of the palate was observed in 3 (5.66%) patients. Healthy mucosa without
any visible inflammatory changes was observed in 23 (34.32%) cases from both groups.
Figure 2 presents the prevalence of denture stomatitis in respective groups under different
treatments and in the control group. Type 1 stomatitis prevails in participants under inhaled
glucocorticoids (19.4%; p = 0.1), bronchodilators (13.4%; p = 0.004) and in control group
(4.5%; p = 0.033). Whereas, there were no significant differences in the distribution of the
other types of prosthetic stomatitis in patients with COPD, dependent on the therapy (p >
0.05).
Figure 2.

Prosthetic stomatitis in patients with chronic obstructive pulmonary disease (COPD) and
control group.

Denture hygiene levels were assessed basing on Ambjörsen's modified plaque index and
rated as poor, and lower in participants with COPD, especially those under inhaled
glucocorticoids and the home oxygen therapy programme, than in the control group (Fig.
3). Denture plaque was detected in 84.9% of COPD participants and 50% control cases. In
37 (55.2%) cases, the plaque build-up was moderate, and it significantly most often
prevailed in participants under bronchodilators (17.9%; p = 0.009). Fourteen COPD cases
(20.9%) presented abundant plaque deposits. A particularly poor oral hygiene was observed
in participants under inhaled glucocorticoids (10.4%; p = 0.61) and oxygen therapy (5.9%;
p = 0.42), but there were no statistic significant differences between the denture plaque
occurrence in groups of patients with COPD and in control group.
Figure 3.

Denture hygiene assessment basing on Ambjörsen's modified plaque index in patients with
chronic obstructive pulmonary disease (COPD) indicating used treatment and control group
[n (%)].

Over 116 bacterial and fungal aerobic strains were isolated and analysed in this study.
Potentially pathogenic microorganisms (PPMs) were isolated from 48 patients with COPD
(90%) and nine participants from the control group (64.3%). The predominant potential
respiratory pathogens were cultured in nine cases from denture swabs patients with COPD,
Enterobacter cloacae (13.4%) and Staphylococcus epidermidis (13.4%). Klebsiella
pneumoniae was detected from 7 (10.4%) swabs. The considerable dominance of Candia
species including 31 C. albicans (46.3%) and seven Candida tropicalis cases (10.4%) was
observed. No significant differences in the frequency of PPMs colonisation to total number
of pathogens in denture plaque were observed between the examined groups (p > 0.05). The
prevalence of respective micro-organisms in the ontocenose of patients with COPD and of
control group participants using removable dentures is presented in Table 1.

Table 1. Type and number of bacterial and fungal strains cultured from direct swabs from
removable dentures in patients with COPD and in healthy subjects from the control group n
(%)
Bacterial species COPD n = 53 (%) Control group n = 14 (%)
1. COPD, chronic obstructive pulmonary disease.
Candida albicans 31 (46.3) 6 (9.0)
Candida glabrata 1 (1.5) –
Candida tropicalis 7 (10.4) 2 (3.0)
Candida parapsilosis 1 (1.5) –
Enterobacter sakazakii 1 (1.5) –
Enterobacter aerogenes 2 (3.0) –
Enterobacter cloacae 9 (13.4) 2 (3.0)
Serratia marcescens 4 (6.0) –
Serratia fonticola 2 (3.0) –
Serratia odorifena 2 (3.0) –
Moraxella lacunata 1 (1.5) 1 (1.5)
Staphylococcus epidermidis 9 (13.4) 2 (3.0)
Staphylococcus cohnii 3 (4.5) –
Staphylococcus aureus 3 (4.5) 1 (1.5)
Klebsiella pneumoniae 7 (10.4) –
Stenotrophomonas maltophila 1 (1.5) –
Pseudomans putida 2 (3.0) –
Pseudomonas aeruginosa 2 (3.0) –
Actinetobacter lwofii 3 (4.5) 2 (3.0)
Escherichia coli 1 (1.5) 1 (1.5)
Physiological flora 3 (4.5) 4 (6.0)
The mycological test revealed after 48 h that yeast-like fungi were present in denture swabs
of 40 (75.5%) patients with COPD and 8 (57%) healthy participants. The strains cultured
from oral mucosal swabs included: C. albicans (31 strains), C. tropicalis (seven strains), C.
glabrata (one strain) and C. parapsilosis (one strain). C. albicans prevailed as often in
participants under bronchodilators as in those under inhaled glucocorticoids. Figure 4
presents the prevalence of respective Candida species in the examined groups. The
variance analysis did not present any significant differences between the groups relative to
strain (p = 0.16) or Candida species prevalence (p = 0.56).

Figure 4.

Candida fungi isolated from denture plaque in patients with chronic obstructive pulmonary
disease (COPD) and healthy subjects from the control group in respective treatment groups
(n).

Discussion
The present study proved patients undergoing chronic treatments were at risk of systemic
infections that could originate in the mouth. Until now, this matter has been given little
attention. Poor oral and denture hygiene is an important factor, predisposing to denture
related mucosal infections[3, 13]. Denture and dental plaque elimination and limiting
bacterial and fungal adhesion to porous acrylic surfaces should be key for limiting general
inflammatory changes. Limitation of this study is fact that patients who are in the control
group are more likely to report at the clinic and are more educated in the topic of hygienic
treatment. They probably better care of oral hygiene than patients with COPD who are less
frequently report on the dental examination. Therefore, the interpretation of the test results
may not be clear. Further studies are needed to clarify this problem. The present study
showed pathogens of the respiratory tract played a significant role in denture plaque build-
up in COPD participants, from whom 16 potentially pathogenic bacterial strains and four
Candida strains were isolated. Comparing with the control group, where six bacterial and
two fungal strains were isolated, it can be safely assumed that denture plaque could be a
potential source of pneumonia and exacerbations of COPD. There has also been an
increasing number of fungal infections, especially Candida. Inhaled glucocorticoid and
home oxygen therapy are particularly responsible for that, as they result in the thinning of
mucosa and mouth dryness, creating opportune conditions for fungal colonisation. The
presence of those pathogens modulates the immune response of the host. Candida
prevalence in patients with COPD amounted to 75.5%, and in the control group to 57%.
Participants under long-term inhaled steroid therapy, who more often have denture
stomatitis, are especially at risk of candidiasis. Batura-Gabryel[14] analysed Candida risk
factors in lung cancer and patients with COPD, indicating those factors were causing acute
recurrent infections, which may more and more often cause death.

It is crucial to make patients with COPD and elderly people using dentures, and also the
medical staffs, aware of how important oral hygiene is and what are the consequences of its
negligence. Liu et al.[15] established there was a higher dental plaque index and a poorer
oral hygiene in patients with frequent exacerbations of COPD. Mechanical denture plaque
removal with brushes, soaps and toothpastes is a generally known method. Scannapieco et
al. suggested oral hygiene in hospitalised patients could be improved using twice a day
0.2% gel with chlorhexidine. It resulted in a lower incidence rate of pneumonia in ICU
patients. Patients should be receiving clear denture and oral hygiene guidelines from the
medical staff and their caregivers[1, 3].

The present study showed that denture plaque in patients with chronic diseases are
colonised by pathogens of the respiratory tract, including: Escherichia coli, Pseudomonas
spp., Klebsiella spp. and Staphylococcus spp. Over 33% of denture isolated pathogens are
part of rod-shaped and gram-negative Enterobacter spp. The isolation of the afore-
mentioned bacteria from denture plaque proves they might become a source of infection of
the respiratory tract, causing exacerbations of COPD. Studies determined microorganism
cells within the biofilm have different phenotypes from free-living cells, and present a
higher drug resistance. Sethi et al.[16] isolated new H. influenzae, M. catarrhalis, and S.
pneumoniae strains from patients with exacerbations of COPD. It is hard to objectively
determine the role of microorganisms in the pathogenesis of chronic diseases (COPD).
Based on the collected data it is not yet possible to precisely confirm the relationship
between inadequate oral hygiene and dental restorations and respiratory tract infections in
COPD. Therefore, further studies examining the role of bacterial and fungal oral flora in
denture stomatitis in patients with COPD, together with determining the interactions
between the different species seem necessary.