SALIVARY PERIODONTAL MARKER BACTERIA...

Romanian Journal of Oral Rehabilitation

Vol. 5, No. 3, July - September 2013

26

SALIVARY PERIODONTAL MARKER BACTERIA RELATED TO

COMMUNITY PERIODONTAL INDEX (CPI) IN NONSMOKERS

VERSUS SMOKERS ROMANIAN ADULTS

Cristina Nuca1*

, Victoria Badea2, Aureliana Caraiane

3

1Ovidius University - Constanta, Romania, Faculty of Dental Medicine,

Department of Preventive Dentistry 2Ovidius University - Constanta, Romania, Faculty of Dental Medicine,

Department of Oral Microbiology 3Ovidius University - Constanta, Romania, Faculty of Dental Medicine,

Department of Oral Rehabilitation

*Corresponding author: Cristina Nuca, Associate Profesor, DMD, PhD

Ovidius University - Constanta, Romania

e-mail: [email protected], phone/fax: 0040 241 66 57 27

ABSTRACT

Background While the relation smoking - periodontal disease is evident, the effect of smoking on oral

microbiota is not fully investigated. Aim of the study To compare the presence of 10 periodontal marker

bacteria in saliva of smokers vs. nonsmokers, in relation with CPI Index. Material and methods A sample of

286 adults (44.05% smokers, 55.94% nonsmokers) participated in a clinical study for collecting unstimulated

saliva (2.5 ml) and recording CPI Index; the salivary samples were analysed for the presence of 10 periodontal

bacteria (classified and scored by pathogenicity), by direct microscopic exam, bacterial cultures and automatic

identification. Ethics approval was obtained. Statistics used SPSS 12. Results CPI and microbiological score

were significant higher in smokers vs. nonsmokers, and correlated with each other (p<0.05). The presence of

periodontal bacteria in the saliva samples was 67.9%, more frequent (p<0.05) in smokers (75.3%) than in

nonsmokers (61.9%); E.corrodens, P.micros, T.denticola, A. actinomycetemcomitans and P.gingivalis were

found more frequently in smokers saliva, the last two being related with CPI; F.nucleatum was found more

frequently in nonsmokers saliva, being related with CPI; the prevalence of P.nigrescens, C.rectus and

P.intermedia did not record significant differences in nonsmokers vs. smokers. Conclusions The smokers saliva

varies considerably from that of non-smokers in terms of periodontal marker bacteria prevalence, this being

related with the periodontal profile assessed by CPI; because the relation between the salivary periodontal

markers bacteria and periodontitis in smokers vs. nonsmokers is essential for the prevention and treatment of

periodontal disease, it needs further long-term studies to elucidate.

Key words: periodontal marker bacteria, smoker, CPI Index, periodontal disease

INTRODUCTION

Periodontal disease is one of the most

common chronic infectious diseases in

humans and is the most prevalent cause of

tooth loss [1]; it is a multifactorial disease of

the tooth supporting structures, elicited by a

microbial biofilm (dental plaque) [2].

The main causes of the development of

periodontal disease are periodontal

pathogens, genetic predisposition within the

immune system, poor oral hygiene, smoking,

systemic diseases and stress; periodontal



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disease is the clinical result of a complex

interaction between the host and plaque

bacteria [3].

Where an active periodontitis is

developing, the body’s defence system plays

a central role. The quantity and virulence of

microorganisms on one hand and host

resistance factors (immune status, genetics

and therefore heredity, as well as the presence

of risk factors) on the other hand are the

primary determinants for the initiation and

progression of periodontal destruction [2].

Normally low concentrations of periodontal

pathogens present even in a healthy sulcus

can be kept in check by an intact immune

system. However, if the defence system is

impaired by a genetic predisposition

(interleukin-1 polymorphism), medication or

smoking, the bacteria can proliferate freely

leading to the manifestation of profound

periodontitis.

The 215 cm2 surface area of the oral cavity

presents numerous surfaces for microbial

colonization, and are continuously bathed in a

bulk fluid, saliva [4]. The teeth provide a

solid, non-shedding surface for the

colonization of potentially pathogenic

bacterial species as well as a wide range of

hostcompatible species [5].

The microorganisms that colonize the oral

surfaces produce biofilms of differing

complexities depending on intraoral location,

genetic background and environmental

factors individual to each subject. As

complex as this microbiota may appear,

approximately 800 species may be detected in

dental plaque and at least 500 in the

periodontal pockets [4,6]. While the majority

of these organisms are commensals subsets of

them are implied in the initiation and

progression of periodontal diseases [7].

Even if it was established the role of some

microorganisms such as Actinobacillus

actinomycetemcomitans (Aa),

Porphyromonas gingivalis (Pg), Bacteroides

forsythus (Bf), Treponema denticola (Td),

Prevotella intermedia (Pi) and Fusobacterium

nucleatum (Fn) in different forms of

periodontal diseases [8], there is no evidence

for any specific pathogen in chronic

periodontitis and therefore it may be

considered as a non-specific bacterial disease

[9].

However, studies of Socransky et al. [4,5,

10, 11, 12, 13], Slots et al. [14, 15, 16] and

others [3,17, 18, 19], have shown that

periodontal disease is caused by a finite set of

bacterial species; only a few of the bacterial

species present in the oral cavity have a high

pathogenic potential that can cause profound

periodontal disease.

A number of possible pathogens have been

suggested on the basis of their association

with disease, animal pathogenicity, virulence

factors, immunological response of the host

to a species, the presence of interacting

bacterial species and the local environment of

the periodontal pocket [5]. Suspected

pathogens (risk markers) of periodontitis

belong to the group of obligatory anaerobic

black-pigmented bacterial species such as

Actinobacillus actinomycetemcomitans,

Porphyromonas gingivalis, Bacteroides

forsythus, Prevotella intermedia, Treponema

denticola and others [3]. According to their

pathogenicity, the first three are considered

very strong pathogenic species, as they

possess a whole range of pathogenic factors,

and their presence in the gingival pocket has

the potential to cause further tooth loss. In

addition to these highly pathogenic species,

other moderate pathogenic species may also

have a pathogenic potential dependent upon

the concentrations in which they are present

(Table 1) [20, 21, 22].

In the same time, Socransky et al. placed

the microorganisms within the oral

microbiota in "complexes" [12, 23]. This

concept emphasized that microorganisms

create their own habitat and interact with each



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other by successive associations till the

appearance of the periodontal disease [8, 12,

23].

Table 1. Pathogenicity of Periodontal Marker Bacteria* [about 20, 21, 22]

Very strong Strong Moderate

Actinobacillus

actinomycetemcomitans (Aa)

Prevotella intermedia

(Pi) Eikenella corrodens (Ec)

Porphyromonas gingivalis (Pg) Treponema denticola

(Td) Prevotella nigrescens (Pn)

Bacteroides forsythus (Bf) Peptostreptococcus micros (Pm)

Fusobacterium nucleatum (Fn)

Campylobacter rectus (Cr)

*the list is not complete

Because all the oral biofilms consist of a

surface needed for the attachment, the biofilm

community itself and the “bulk fluid” (saliva

and/or gingival crevicular fluid) that passes

over the biofilm [4], the microbiological

diagnosis of the periodontal disease can be

made by analyzing dental plaque (sub- and

supragingival), and also by analyzing the

salivary microbial composition.

As a diagnostic fluid, saliva offers some

advantages: non–invasive and easy collection

procedure and high sensitivity and correlation

with levels in blood for detection of many

oral and systemic diseases [24, 25, 26, 27].

Saliva contains locally-produced microbial

and host response mediators, as well as

systemic (serum) markers that may be used in

the diagnosis of periodontal disease [28, 29,

30].

Based on the literature [28, 31], salivary

markers that have been studied as potential

diagnostic tests for periodontal disease

include proteins of host origin (i.e., enzymes,

immunoglobulins), phenotypic markers, host

cells, hormones (cortisol), ions and volatile

compounds and also bacteria and bacterial

products.

Determination of the numbers of a given

bacterial species in non-stimulated saliva may

indicate whether it is actively growing in

plaque and microbiological tests on the oral

flora should be used to monitor the oral health

[32], especially since the comparison of

microbial composition of biofilms on teeth,

soft tissues and saliva [17] showed a very

high similarity regarding the mean species

proportion.

As we enter the era of genomic medicine,

the salivary analysis plays an increasingly

important role in the detection and monitoring

of oral and systemic diseases [33].

Against this background, the aim of this

study is to compare the presence of 10

periodontopathic bacteria, classified by their

pathogenicity, in whole saliva of smokers vs.

nonsmokers, in relation with their periodontal

status assessed by CPI Index.

MATERIAL AND METHODS

A. Study population and sample:

The study subjects consisted of 286

individuals from Constanta District (6%

sampling error; 95% confidence level), with

age range 35-44 years (mean 40± 4 years),

including 126 (44.05%) smokers and 160

(55.94%) nonsmokers who had not received

antibiotics within the previous three months.

B. Generation and collection of the saliva

samples

Total unstimulated saliva samples

(minimum 2.5 ml) were collected from all

subjects using a standard method (passive



29

collection in sterile containers). The saliva

samples were transported immediately after

collection to the microbiology laboratory

(Ovidius University, Faculty of Dental

Medicine), in order to carry out the

microbiological examination.

C. Clinical examination

The clinical examination of the subjects

was made by two trained and calibrated

examiners, recording the CPI Index using

World Health Organization (W.H.O.) 1997

criteria [34].

The clinical examination was carried out

in the selected family offices, using plain

mouth mirrors, W.H.O. (621 type)

periodontal probes and sterile gloves, under

artificial optimal light and respecting the

usual infection-control protocols. No

instruction in tooth brushing or oral

prevention was given to the participants prior

to the start of the study. Each examination

was performed in the morning (between 10

and 12 a.m.).

All the CPI Index teeth (or all the

remainder teeth in a sextant where there is no

index tooth) were examined at 6 sites, and the

highest score was recorded; each sextant was

given a CPI score (0 - healthy; 1 - gingival

bleeding; 2 - calculus; 3 - shallow pockets; 4 -

deep pockets) and the maximum CPI was

recorded as the individual’s Index.

D. Microbiological testing of the saliva

samples

The saliva samples were

microbiological tested in order to identify the

following 10 anaerobic bacteria (periodontal

marker bacteria):

1. Eikenella corrodens (Ec),

2. Prevotella nigrescens (Pn),

3. Peptostreptococcus micros (Pm),

4. Fusobacterium nucleatum (Fn),

5. Campylobacter rectus (Cr),

6. Prevotella intermedia (Pi),

7. Treponema denticola (Td),

8. Actinobacillus actinomycetemcomitans

(Aa),

9. Porphyromonas gingivalis (Pg),

10. Bacteroides forsythus (Bf).

For each sample, the bacteriological exam

followed the next stages:

I) direct microscopic exam;

II) bacterial cultures;

III) biochemical automatic identification

using mini API® system.

I) The direct microscopic exam was made by:

direct optical examination made on fresh

preparation between blade and slide and also

on simple and Gram stained smear; this exam

allowed to identify the morphological

features of the targeted bacteria;

dark-field microscopy - this method

reveals only the morphotypes of bacteria, i.e.

the shape of bacteria and their motility, but

does not permit any identification of bacterial

classifications or species; it permits

differentiation between inactive and active

samples; if the sample reveals primarily cocci

and non-motile rods, it is an indication of

only few active pathogens; if the field

exhibits numerous motile bacteria (e.g., rods

and spirochetes) it is an indication of a

potentially pathogenic flora.

II) Bacterial cultures :

selective culture media were used, as

follows:

- Schaedler agar with neomycin,

vancomycin and 5% ram blood for

A.actinomycetemcomitans (Aa),

P.intermedia (Pi), P.nigrescens (Pn)

and F.nucleatum (Fn) identification;

- Schaeldler agar with K vitamin for

B.forsythus (Bf); Columbia agar with

5% ram blood for P.gingivalis (Pg),

C.rectus (Cr), E.corrodens (Ec) and

T. denticola (Td) identification.

the biological products seeded on these

culture media were incubated in



30

microaerophilic conditions at 36°C±20C for 5

days in order to identify the facultative

anaerobic species (Aa) and in strictly

anaerobic conditions (Jar anaerobic system

and Anaerocult® A sachets containing

components which chemically bind oxygen,

creating an oxygen-free anaerobic milieu and

a CO2 atmosphere) at 36°C (±20C) for 7-10

days for the obligatory anaerobic bacteria

(Merck KGaA, Germany) [35].

the bacteria colonies developed after

incubation were verified by direct

microscopic exam made on Gram stained

smear and were then transplanted for

achieving pure cultures and for further

identification in API system.

III) Biochemical automatic identification

the identification of bacterial species was

made using an API bioMérieux system and

API® 20 A strips (bioMérieux® SA, France),

which allow the rapid identification of

anaerobic bacteria using 21 biochemical tests

(on the basis of 20 dehydrated and

miniaturized culture medium) [36];

the preparation of the bacterial suspension

was made with the verification of the optimal

density, using a densimat; the optimal

turbidity for anaerobic bacteria identification

was calculated at 3 Mc Farland units;

the API® 20 A were inoculated with

bacterial suspension and incubated at 360C

±20C for 24 - 48 hours in an anaerobic jar;

as a result of metabolic processes, in the

incubation period spontaneous and chemical

induces by adding supplementary reagents as

XYL (xylose), BCP (1-bromo-3-

chloropropane) and EHR (Ehrlich’s Reagent),

colour changes took place;

reading and interpretation of the results

(after the incubation period) was made using

the corresponding tables (identification

profiles) and was then confirmed using the

identification soft provided by mini API®

expert system.

Ethics approval

Ethical permission to conduct the study

was given by the Professional Ethical

Committee of Ovidius University, Constanta.

Free-written informed consent (including

patient information on the aim and methods

of the study) was obtained from all the

participants. Participation was optional, and

the time for thinking (express the consent or

refusal) was 48 hours.

Statistical analyses

These were made using SPSS 12 for

Windows. Chi-square test was used for

testing intra-group variation. ANOVA was

used for testing the between-groups variation.

Spearman and Pearson coefficients were used

for measuring the correlation/association

between variables. U-statistic (Wilks’

Lambda) was used for the multivariate

analysis.

RESULTS

A. The CPI Index

The mean value of CPI Index was

2.45±1.07, significant higher in smokers

(2.71±1.00) vs. nonsmokers (2.26±1.09)

(p=0.000; ANOVA).

The frequencies of CPI values in

nonsmokers and smokers are shown in Table

2.

Table 2. The CPI Index values in nonsmokers vs. smokers

CPI Index nonsmokers smokers

Count % Count %

0 (healthy) 18 11.3% 6 4.8%

1 (gingival bleeding) 6 3.8% 2 1.6%



31

2 (calculus) 73 45.6% 45 35.7%

3 (shallow pockets) 43 26.9% 43 34.1%

4 (deep pockets) 20 12.5% 30 23.8%

Total 160 100% 126 100%

p<0.05 (Chi-square statistics)

B. The microbiological score

The targeted identified bacteria were

classified according to their pathogenicity

(Table 1) in the following categories [20, 21,

22]:

moderate pathogenic species: Eikenella

corrodens (Ec), Prevotella nigrescens

(Pn), Peptostreptococcus micros (Pm),

Fusobacterium nucleatum (Fn),

Campylobacter rectus (Cr).

strong pathogenic species: Prevotella

intermedia (Pi), Treponema denticola

(Td).

very strong pathogenic species:

Actinobacillus actinomycetemcomitans

(Aa), Porphyromonas gingivalis (Pg),

Bacteroides forsythus (Bf).

According to this classification, in order

to statistically analyze the results of the study,

each subject was given a microbiological

score, corresponding with the highest

pathogenic species category of bacteria

identified in its individual saliva sample, as

follows:

0 - the microbiological exam did not

reveal the presence of any bacterial

species;

1 - the microbiological exam revealed

other species than those targeted in the

study;


moderately pathogenic anaerobic species

± other species;


strongly pathogenic anaerobic species ±

other species;


very strongly pathogenic anaerobic

species ± other species.

The targeted periodontal bacteria were

found in 67.9% (n=194) of the saliva

samples, more frequently (p<0.05; Chi-

square statistics) in smokers (75.3%) than in

nonsmokers (61.9%); 32.1% of samples,

significant more (p<0.05; Chi-square

statistics) in nonsmokers (38.2%) than in

smokers (24.6%), could not reveal the

presence of periodontal marker bacteria

(Table 3).

Table 3. The microbiological score values in nonsmokers vs. smokers

Microbiological

score

nonsmokers smokers Total

n % n % n %

no targeted

bacteria

0 30 61

18.8 38.2

11 31

8.7 24.6

41 92

14.3 32.1

1 31 19.4 20 15.9 51 17.8

with targeted

bacteria

2 35

99

21.9

61.9

27

95

21.4

75.3

62

194

21.7

67.9 3 25 15.6 28 22.2 53 18.5

4 39 24.4 40 31.7 79 27.6

Total 160 100.0 126 100.0 286

100.0 p<0.05 (Chi-square statistics)

The frequencies of the microbiological scores in nonsmokers vs. smokers and in the



32

entire sample are shown in Table 3; the mean

value of the general microbiological score

was 2.27±1.40, significant higher in smokers

(2.52±1.31) vs. nonsmokers (2.07±1.44)

(p=0.007; ANOVA).

Some of the bacterial cultures obtained

after the first inoculation and API® 20A

strips with the final results are presented in

figures 1-4.

Figure 1. Prevotella (above) and Bacteroides

and Porphyromonas (down) species Figure 2. Prevotella and Porfiromonas

species.

Figure 3. Actinomices and Bacteroides

species identified on API® 20A strips Figure 4. Prevotella and Bacteroides

species identified on API® 20A strips

C. The relationship between individual’s

microbiological score and CPI Index in

smokers/nonsmokers

The analysis of the possible relationship

between individual’s microbiological score

and CPI Index showed that there is a positive

correlation (Spearman coefficient=0.575;

p.=0.000) between the general microbial

score and CPI Index in the entire study

sample, for nonsmokers (ns) and also for

smokers (sm) (Table 4).

D. The periodontal anaerobic bacteria

identification

The comparison of the 10 periodontal

marker bacteria identification in the saliva of

nonsmokers vs. smokers was made only on

subjects with microbiologic scores 2, 3 and 4,

were at least 1 of the targeted bacteria was

identified; the subjects with microbiological

score 0 (n=41; 14.3%; Table 3) and 1 (n=51;

17.8%; Table 3) were excluded from this

analysis.

The new study sample comprised 194

subjects (99 nonsmokers and 95 smokers) and

the frequency of each targeted bacteria

identification is shown in Table 5.



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Table 4. The relationship between microbiological score and CPI in nonsmokers/smokers

Smoker category CPI Index microbiological score Sperman correlation

(value/sig.) 0 1 2 3 4

ns

(n=160)

0 11 3 3 1 0

0.604/0.000

1 2 1 3 0 0

2 17 20 16 10 10

3 0 7 11 12 13

4 0 0 2 2 16

sm

(n=126)

0 3 1 0 1 1

0.484/0.000

1 1 1 0 0 0

2 6 14 12 7 6

3 0 3 9 14 17

4 1 1 6 6 16

Table 5. Frequency of bacteria identification in nonsmokers vs. smokers

Bacteria

Bacteria identification (n, %)

p (ANOVA) Total frequency (n, %)

nonsmokers smokers

Ec 7 (7.1%) 16 (16.8%) 0.035 23 (11.9%)

Pn 9 (9.1%) 0 (3.2%) 0.087 12 (6.2%)

Pm 10 (10.1%) 22 (23.2%) 0.014 32 (16.5%)

Fn 23 (23.2%) 11 (11.6%) 0.033 34 (17.5%)

Cr 16 (16.2%) 25 (26.3) 0.084 41 (21.1%)

Pi 28 (28.3%) 17 (17.9%) 0.087 45 (23.2%)

Td 14 (14.1%) 29 (30.5%) 0.006 43 (22.2%)

Aa 17 (17.2%) 31 (32.6%) 0.012 48 (24.7%)

Pg 13 (13.1%) 25 (26.3%) 0.021 38 (19.6%)

Bf 14 (14.1%) 12 (12.6%) 0.759 26 (13.4%)

E. corrodens, P.micros, T.denticola,

A.actinomycetemcomitans and P.gingivalis

were found more frequently in the smokers’s

saliva, and F.nucleatum was found more

frequently in the nonsmokers saliva (p<0.05;

ANOVA).

The prevalence P.nigrescens, C.rectus and

P.intermedia din not record significant

differences between nonsmokers and smokers

(p>0.05).

E. The relationship between the

periodontal marker bacteria and CPI Index

in nonsmokers/smokers

CPI Index was significant correlated

(Spearman coefficient) with F.nucleatum,

P.gingivalis and A.actinomycetemcomitans

presence in nonsmokers and with

F.nucleatum, P.gingivalis and B.forsythus

presence in smokers (Table 6).

F. The association between the

periodontal markers bacteria in

nonsmokers/smokers and their relationship



34

with CPI Index

The associations (Pearson coefficient)

between the targeted bacteria in

nonsmokers/smokers are shown in Table 7.

The multivariate analysis of the linear general

model showed that only some of these

bacteria associations are related (U-statistic)

with CPI Index.

Table 6. The relationship between bacteria identification and CPI in nonsmokers/smokers

Bacteria

category

Smoker

category

Bacteria

identification

CPI Index p

(Spearman) 0 1 2 3 4

Ec

ns no 4 3 34 31 20

p=0.853 yes 0 0 2 5 0

sm no 2 - 22 33 22

p=0.278 yes 0 - 3 7 6

Pn

ns

no 3 2 34 32 19 p=0.482

yes 1 1 2 4 1

sm no 1 - 24 39 28

p=0.070 yes 1 - 1 1 0

Pm

ns

no 4 2 33 30 20 p=0.554

yes 0 1 3 6 0

sm no 1 - 20 31 21

p=0.863 yes 1 - 5 9 7

Fn

ns

no 2 1 22 32 19 p=0.000

yes 2 2 14 4 1

sm no 2 - 19 36 27

p=0.037 yes 0 - 6 4 1

Cr

ns no 3 3 29 32 16

p=0.881 yes 1 0 7 4 4

sm no 1 - 22 27 20

p=0.279 yes 1 - 3 13 8

Pi

ns no 4 3 25 26 13

p=0.319 yes 0 0 11 10 7

sm no 1 - 21 32 24

p=0.619 yes 1 - 4 8 4

Td

ns no 3 3 34 29 16

p=0.133 yes 1 0 2 7 4

sm no 1 - 20 27 18

p=0.317 yes 1 - 5 13 10

Aa

ns no 4 3 33 28 14

p=0.012 yes 0 0 3 8 6

sm no 1 - 18 29 16

p=0.317 yes 1 - 7 11 12



35

Pg

ns no 4 3 33 33 13

p=0.009 yes 0 0 3 3 7

sm no 2 - 24 26 18

p=0.007 yes 0 - 1 14 10

Bf

ns no 4 3 31 32 15

p=0.201 yes 0 0 5 4 5

sm no 2 - 25 34 22

p=0.017 yes 0 - 0 6 6

Table 7. Bacteria associations in nonsmokers/smokers

Smoker category Bacteria associations p

(Pearson)

p

(U-statistic)

nonsmokers

Pn and Pi 0.049 p>0.05

Pm and Pi 0.019 p>0.05

Fn and Pg 0.034 p=0.000

Fn and Bf 0.026 p>0.05

Cr and Bf 0.032 p>0.05

smokers

Ec and Cr 0.046 p>0.05

Ec and Aa 0.001 p>0.05

Ec and Pg 0.000 p=0.038

Fn and Aa 0.014 p=0.001

Fn and Pg 0.035 p>0.05

Cr and Pi 0.035 p>0.05

Cr and Aa 0.039 p>0.05

Cr and Bf 0.027 p>0.05

Pi and Pg 0.032 p>0.05

Td and Aa 0.034 p>0.05

Aa and Pg 0.003 p=0.025

Aa and Bf 0.007 p>0.05

Pg and Bf 0.001 p=0.018

DISCUSSIONS

Numerous studies have examined the

relationship between smoking and periodontal

diseases, showing a higher level of

periodontal disease in terms of increased

alveolar bone loss, less bleeding on probing,

increased number of deep periodontal pockets

and greater attachment level loss in current

smokers than in non smokers (past and never

smokers) [4, 37-44].

While the strong relationship between

smoking and severity of periodontal disease is

evident, the effect of smoking on the

composition of oral microbiota is less clear.

Numerous studies were made in terms of

microbial composition of the subgingival

plaque in smokers vs. nonsmokers, with

various results.

Some studies showed that cigarette

smoking has little impact on subgingival

plaque composition. Preber et al. [45] showed

that counts of A.actinomycetemcomitans,



36

P.gingivalis, and P.intermedia were not

significantly different in smokers vs.

nonsmokers in deep pockets on adult

periodontitis subjects; Stoltenberg et al. [46]

found no significant differences between

smokers and nonsmokers in the prevalence of

A.actinomycetemcomitans, P.gingivalis,

P.intermedia, E.corrodens, and F.nucleatum

and Lie et al. [47] found no difference in

smokers vs. nonsmokers in the counts of nine

subgingival species in adults with gingivitis.

There are also other studies that have

found differences between the subgingival

microbiota of smokers and nonsmokers.

The study of Eggert et al. [48]

demonstrated that P.gingivalis, P. intermedia,

and A. actinomycetemcomitans were found

more frequently in the shallow pockets of

smokers than in similar sites in nonsmokers.

Kamma et al. [49] found that proportions

and/or prevalence of P.micros, C.concisus, B.

forsythus, C.rectus, C.gracilis, Selenomonas

sputigena, and P.gingivalis were significantly

elevated in smokers, whereas Spreptococcus

intermedius, A.naeslundii, Actynomices

israelii, and Eubacterium lentum were

significantly higher in nonsmokers. Zambon

et al. [50] found that smokers had

significantly higher levels of B..forsythus than

nonsmokers.

Data from the study of Haffajee and

Socransky [51] indicated that, even if there

were no significant differences in levels and

proportions of 29 test species from

subgingival microbiota in different smoking

groups, the prevalence of several orange,

E.nodatum, F.nucleatum, P. intermedia, P.

micros, and P. nigrescens, as well as all three

red complex species P. gingivalis, T.

forsythia, and T. denticola was significantly

greater in smokers than in non smokers; the

difference in prevalence of subgingival

species among smoking groups was

particularly marked in deep pockets.

The relation between dental plaque (supra-

and subgingival) and salivary microbiota is

also well documented. A study of Umeda et

al. [52] compared the presence of 6

periodontopathic bacteria in whole saliva and

subgingival plaque. Their results indicated

that whole saliva is superior to pooled

periodontal pocket samples to detect P.

gingivalis, P. intermedia, P. nigrescens, and

T. denticola in the oral cavity; however, little

agreement between samples was found for A.

actinomycetemcomitans and B. forsythus. The

comparison made by Mager et al. [17] on

three oral clusters of biofilms (plaque, soft

tissues and saliva) showed that there is an

over 80% similarity between the mean

species proportion from saliva and dental

plaque and the study of Rodrigues de Araújo

Estrela [53] showed no differences for the

prevalence of P.intermedia and P.gingivalis

in saliva, other oral sites (tongue dorsum,

buccal mucosa) and dental plaque (supra- and

subgingival).

Even if these studies demonstrated the

usefulness of salivary microbial composition

analysis for periodontal health, the studies

regarding the relation between salivary

microbiota, smoking and periodontal health

are not so numerous.

Our study found that the presence of the

targeted periodontal bacteria in the saliva

samples was by 67.9%, more frequent in

smokers (75.3%) than in nonsmokers

(61.9%);, and the general mcrobiological

score (the presence of the targeted bacteria)

was correlated with the periodontal status

assessed by CPI. In the same time, the present

results showed that E.corrodens, P.micros,

T.denticola, A. actinomycetemcomitans and

P.gingivalis were found more frequently in

the smokers saliva, the last two being related

with CPI; F.nucleatum was found more

frequently in the nonsmokers saliva, being

also related with CPI. The frequency of

P.nigrescens, C.rectus and P.intermedia

identification din not record significant



37

differences between nonsmokers and

smokers.

In a study regarding the salivary

periodontitis bacteria, Darout et al. [54]

showed that a high percentage of the subjects

had detectable levels of several bacterial

species in saliva; between 12% and 16% of

the subjects showed high salivary levels of

the periodontitis-associated bacteria A.

actinomycetemcomitans, P. melaninogenica,

P. intermedia, C. rectus and E. corrodens,

whereas only two (3.6%) and four (7.1%)

subjects had high levels of P. gingivalis and

F. nucleatum, respectively. There were

significantly higher levels of A.

actinomycetemcomitans, C. rectus, P. micros,

and significantly lower levels of P.

intermedia, F. nucleatum, E. corrodens in the

smokers than in the nonsmokers group.

Comparing with this study, our study

showed higher rates of all these evaluated

bacteria (between 11.9 and 24.7%) and a very

high presence of the targeted periodontal

bacteria in saliva; regarding the bacteria

prevalence in smokers vs. nonsmokers, our

results are in agreement with the results of the

cited study, showing that the periodontal

bacteria presence was more frequent in

smokers than in nonsmokers. Comparing with

the study of Rodrigues de Araújo Estrela et

al. [53], our study found similar results

regarding the prevalence of P. intermedia and

P. gingivalis in saliva.

Regarding the differences of bacterial

presence in smokers vs. nonsmokers, the

present study is in accordance with the study

of Darout et al. [54] in terms of a higher

frequency of A. actinomycetemcomitans and

P. micros and a lower frequency of F.

nucleatum in smokers.

The study made by Umeda et al. [55]

found that past smokers had a decreased risk

of harboring A. actinomycetemcomitans in

saliva, while current smokers had an

increased risk of harboring T.denticola,

although the risk of colonization by P.

intermedia and P.nigrescens did not differ

among smoking groups. Our study found

similar results regarding the absence of

differences between smokers and nonsmokers

in terms of P. intermedia and P.nigrescens

prevalence, and also regarding the higher

presence of A. actinomycetemcomitans and

T.denticola in smokers saliva.

The results of the present study regarding

the associations between bacteria and their

relationship with CPI Index are in agreement

with the studies made by Haffajee and

Socransky [51], showing significant

associations between all the bacteria of the

red complex (P.gingivalis, B.forsythus -

intensely and T.denticola - strongly

pathogenic) and also associations of Aa

(purple complex, intensely pathogenic) with

some of the red complex bacteria

(P.gingivalis and B.forsythus - intensely

pathogenic) in smokers; the main associations

regard the highly and strongly pathogenic

bacteria and are related with CPI. In

nonsmokers, the red complex bacteria

(P.gingivalis and B.forsythus, intensely

pathogenic) are associated with bacteria with

less pathogenic potential (F.nucleatum -

orange complex, and C.rectus - green

complex, both moderately pathogenic) and

the main associations regard the strongly

(P.intermedia) and the moderately bacteria

(P.micros, F.nucleatum), the most being not

related with CPI. There are also two

associations present in both smokers and

nonsmokers, between intensely and

moderately pathogenic bacteria (as

F.nucleatum with P.gingivalis, C.rectus with

B.forsythus), depending on CPI.

We can conclude that the present study is

enrolling in the actual studies regarding the

salivary microbial profile of smokers vs.

nonsmokers in relation with the periodontal

status, showing that smokers saliva varies

considerably from that of non-smokers in



38

terms of significant differences in the

prevalence and abundance of periodontal

marker bacteria, these being related with the

periodontal profile assessed by CPI Index.

Because of the increasing prevalence of

both smoking habit and periodontal disease in

adults but especially in young people at a

global level, long-term longitudinal studies

are required to establish the relationship

between the salivary periodontal markers

bacteria and prevalence and progression of

periodontitis in smokers vs. nonsmokers,

saliva being a readily available and cost-

effective diagnosis fluid for periodontal

disease assessment in large populations.

Acknowledgements

The authors would like to thank to all subjects for their unconditional co-operation and

support.

This work was supported by CNCSIS – UEFISCSU, project number PNII – IDEAS

1216/2008 - “Studies for evaluation of cotinine and other biomarkers in the oral fluids, as a base

for the development of a non-invasive diagnosis method and a prognosis model of the periodontal

disease in smokers”.

Statement of conflicts of interest

The authors of this article are not aware of any conflicts of interests regarding this study.

Abbreviations:

CPI - Community Periodontal Index

WHO - World Health Organization

SPSS - Statistical Package for the Social Sciences

versus - vs.

Eikenella corrodens - E. corrodens / Ec

Prevotella nigrescens - P. nigrescens / Pn

Peptostreptococcus micros - P.micros / Pm

Fusobacterium nucleatum - F.nucleatum / Fn

Campylobacter rectus - C.rectus / Cr

Prevotella intermedia - P.intermedia / Pi

Treponema denticola - T.denticola / Td

Actinobacillus actinomycetemcomitans - A.actinomycetemcomitans / Aa

Porphyromonas gingivalis - P.gingivalis / Pg

Bacteroides forsythus - B.forsythus / Bf

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