1225

Association Between Cigarette

Smoking. Bacterial Pathogens, and
Periodontal Status
Jill L.

Stoltenberg, Joy B. Osborn, * Bruce L. Pihlstrom, Mark C. Herzberg,

Dorothe M. Aeppli, * Larry F. Wolff, and George E. Fischer*
*

The

1) an association exists between

disease
after
cigarette smoking
signs periodontal
controlling for the confounding
variables of age, sex, plaque, and calculus; 2) the prevalence of 5 bacteria commonly
associated with periodontal disease differs between smokers and non-smokers; and 3)
the presence of any of these bacteria or smoking are associated with a mean proximal
posterior probing depth > 3.5 mm. Plaque, calculus, gingivitis, and probing depth were
measured at the proximal surfaces of all teeth in one randomly selected posterior dental
sextant in 615 adults. Subgingival plaque was sampled from the same sites and assayed
for the presence of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans,
Prevotella intermedia, Eikenella corrodens, and Fusobacterium nucleatum. A subsample
of non-smokers (n =, 126), who were similar to smokers (n
63) with respect to age,
sex, plaque, and calculus, was randomly drawn from the original sample. These two
groups were then compared on the basis of clinical and microbial parameters. The results
indicated that the odds of having a mean probing depth > 3.5 mm were 5 times greater
for smokers than the non-smoker subsample (odds ratio
5.3; 95% CI 2.0 to 13.8).
No statistically significant difference in the prevalence of any of the bacteria was found
between smokers and the non-smoker subsample. Based on logistic regression analyses
of each of the 5 bacteria and smoking, mean probing depth > 3.5 mm was significantly
associated with the presence of A. actinomycetemcomitans, P. intermedia, E. corrodens,
and smoking (P < 0.05). For this sample of medically healthy, middle-aged adults, it
is concluded that cigarette smoking is a stronger risk indicator for the presence of a mean
posterior proximal probing depth > 3.5 mm than any of the 5 bacteria. / Periodontol
purposes of this study were to determine if:

and

of

1993;64:1225-1230.

Key Words: Periodontal disease/microbiology; periodontal diseases/etiology; smoking/

adverse effects.

Clinical findings related to smoking and periodontal dishave been well-documented.1"28 Alveolar bone
loss,2"7 tooth mobility,7 increased probing depth,7 9 and
tooth loss6"7'10"12 have been reported to be more severe
in smokers than non-smokers. Certain types of periodontal disease, such as refractory Periodontitis13 and acute
necrotizing ulcerative gingivitis,14 have also been associated with cigarette smoking. Tobacco smoke and its
water soluble components have been shown to alter host
resistance, and response and repair mechanisms by adversely affecting normal polymorphonuclear leukocyte
function.13'15"18 Other studies have confirmed the relaease

*Clinical Research Center for Periodontal Diseases,

nesota, School of Dentistry, Minneapolis, MN.
+Group Health, Incorporated, Minneapolis, MN.

University of

Min-

tionship between smoking and periodontal disease by

identifying20 smoking as a risk indicator for increased probing
depth8-19 and loss of periodontal attachment.20 23
The greater severity of disease in smokers appears to be
independent of factors such as age, race, sex, income, education, and oral hygiene practices.24 While the effect of
plaque accumulation in smokers and non-smokers has been
controlled in many studies,8'25 26 few reports have con-

trolled for the influence of calculus.4 Yet, calculus has been

reported to be more prevalent in smokers than non-

smokers.7'26"28

Few studies have examined the oral microbial flora of

smokers and non-smokers. In vitro exposure of bacteria to
cigarette smoke resulted in a marked decrease in the numbers of viable bacteria.29 31 Gram positive species have been
reported to be both more30 and less31 resistant to cigarette

1226

CIGARETTE

SMOKING,

BACTERIAL

J Periodontol
December 1993

PATHOGENS, AND PERIODONTAL STATUS

smoke than Gram-negative bacteria. No significant differin the prevalence of various plaque bacteria was found
between the cultured dental plaque of smokers and
ence

non-smokers.32"33

The purposes of this study were to determine if: 1) an

association exists between cigarette smoking and signs of
periodontal disease after controlling for the confounding
variables of age, sex, plaque, and calculus; 2) the prevalence of 5 bacteria commonly associated with periodontal
disease differs between cigarette smokers and non-smokers;
and 3) the presence of any of these bacteria and/or smoking
are associated with mean proximal posterior probing depth
> 3.5 mm.
METHODS AND MATERIALS

Study Population
The sample for the present study has been described elsewhere in more detail.34 Briefly, it consisted of a subsample
of 1,090 healthy adults who attended a large health maintenance organization in the Minneapolis-St. Paul area. Data
were available for all bacteria under investigation at each
clinically measured site in 615 of the 1,090 subjects. After
preliminary analysis, a subsample of non-smokers (n
126), who were similar to smokers (n 63) with respect
to age, sex, plaque, and calculus, was randomly drawn
from these 615 subjects. Smoking habits were self-reported.
Subjects who indicated use of other forms of tobacco (cigar,
pipe, or smokeless tobacco) were excluded from the analyses. The protocol for all procedures was approved by the
University of Minnesota Institutional Review Committee
for Human Subjects. Informed consent was obtained as approved by this committee.
=

Plaque Sampling and Clinical Documentation

subject, the proximal surfaces (mesio-buccal, raesio-lingual, disto-lingual, disto-buccal) of premolare and
molars in a randomly selected posterior sextant were sampled for subgingival plaque and documented clinically by
In each

trained and calibrated examiners.35"36 Block randomization,

stratified by age and sex, was achieved by a random number
generator. Third molars were excluded. After clinically
scoring37 and removing supragingival plaque, subgingival
plaque samples were obtained using Gracey curets in a manner
similar to previous studies.38"39 Buccal and lingual samples
from each proximal surface were pooled in 250 ml sterile
phosphate buffered saline. This collection technique yielded
one mesial and one distal plaque sample per tooth for subsequent immunoassay. Plaque samples were transferred to
the immunology laboratory at the University of Minnesota
School of Dentistry and stored at -20C until assayed. Following plaque collection, a calculus index (CI),40 gingival
index (Gl)41 probing depth (PD), bleeding upon probing,42
and relative attachment level were recorded at each site.

Two automated probes,* were used to obtain probing depth

and relative attachment level at each site as earlier
described.34 364344 Since relative attachment level recorded
with the disk probe is only valid for documenting change
over time, attachment level measurements were not included in this cross-sectional study.

Immunoassay for Evaluating Bacteria in Plaque

For each

subject, mesial and distal plaque samples were

separately assayed for P. gingivalis, A. actinomycetemcomitans, P. intermedia, E. corrodens, and F. nucleatum by a
semi-quantitative bacterial concentration fluorescence immunoassay.45 Therefore, for the total sample of 615 subjects, over 4,000 separate assays were run for each of these
bacteria yielding a total of more than 20,000 immunoassays. Based on comparison to standard controls, each sample was considered positive for a specific bacterium if the
relative number of bacterial cells was greater than approximately 5 103 bacteria per plaque sample. A subject was
considered positive for the bacterium if it was detected in
at least one of the proximal samples.

Statistical Methods
As a preliminary analysis, Pearson correlations were computed on the entire study population (N 615) to examine
associations between clinical variables. To control for confounding clinical variables between the number of smokers
(n 63) and non-smokers (n 552), a subsample of nonsmokers, frequency matched 2:1 with smokers for the standard variables of age and sex, and the confounding clinical
variables of plaque and calculus, was randomly drawn. The
2:1 ratio was selected in order to maximize statistical power
within the constraints of matching. Summary data for smokers
and the subsample of non-smokers were calculated to provide a clinical description of disease prevalence and severity
in these subjects. Differences in mean probing depth were
tested using a 2 sample r-test. Comparisons between cate=

gorized variables were evaluated by chi-square statistics,

which included median tests. An odds ratio was calculated
for the presence of more severe periodontal disease defined
as mean probing depth > 3.5mm. Prevalence data for each
of the 5 bacterial species were determined for smokers (n
63) and the non-smoker subsample (n 126). Logistic
regression analyses were used to determine if any of the
bacteria or smoking were statistically significant indicators
of mean posterior proximal probing depth > 3.5 mm. Computations were carried out through the use of computer software program. In all statistical tests, a probability value
< 0.05 was considered statistically significant. No
of
adjustment for multiple testing was introduced.
=

Florida Probe and Florida Disk Probe, Florida Probe

Corp., Gainesville,

FL.

8SPSS/PC+ TM4.0 Statistical Package for the Social Sciences, Inc., Chicago, IL.

Volume 64
Number 12

STOLTENBERG, OSBORN, PIHLSTROM, HERZBERG, AEPPLI, WOLFF, FISCHER

RESULTS
The age of

<

0.001)

3.5 mm are presented in Table 2. Fifteen

the
of
smokers
had a mean probing depth > 3.5
(23.8%)
mm compared to 7 (5.6%) of the non-smoker subsample.
The odds of smokers having a mean PD > 3.5 mm were
5.3 times greater than for the non-smoker subsample. There
was no statistically significant difference between smokers
and the non-smoker subsample in the prevalence of P. gingivalis, A. actinomycetemcomitans, P. intermedia, E. corrodens, or F. nucleatum (P > 0.05). Each bacterium was
identified in 40 to 50% of both smokers and the non-smoker
>

subjects in the population sample (

615)
from
28 to 73 years (
51.0; sd
10.5). Apranged
proximately two-thirds (66.7%) of the subjects were female. Sixty-three individuals (10.2%) reported smoking
cigarettes. Among the subjects who reported smoking, 66.7%
(n 42) were female. Cigarette smokers were slightly
48.1 years; sd
10.3) than non-smokers
younger (
the
sd
with
( 51.3;
10.5)
majority of smokers (81.0%)
the
of
and
59. Smokers reported an
between
35
being
ages
of
16.6
(sd 11.8) cigarettes smoked daily. Past
average
and/or
duration
of smoking by subjects were not
history
known. Data analyses of the entire sample (n
615) revealed no significant association between the clinical variables of mean gingival index > 0 and probing depth or
mean bleeding upon probing > 0 and probing depth. However, due to the large sample size, weak associations between mean plaque and probing depth (r
0.13; <
mean
and
calculus
and
0.001)
probing depth (r 0.14;
were

statistically significant.

Since the smokers (n

63) and non-smoker subsample
(n 126) were matched for age, sex, plaque, and calculus,
the groups were similar for these parameters. The two groups
were matched exactly with respect to sex (female/male
66.7%/33.3%), and were close in mean age (smokers: 48.1
10.3; non-smoker subsample: 49.0 years; sd
years; sd
10.2). Seventy-three percent of the smokers and nonsmoker subsample had no detectable calculus (CI < 1) at
any site and 27.0% had subgingival calculus (CI > 1) at
one or more sites. Smokers and the subsample of nonsmokers were similarly matched for plaque with 60.3% of
both groups having had a plaque index > 1 at less than 10%
of the sites.
There were clinically small, but statistically significant,
differences between smokers and non-smoker subsample in
the number of sites measured and mean proximal probing
depth per subject. Out of a possible total of 16 sites, smokers had an average of 0.8 fewer sites measured than the
1.9 and 15.2 sites,
non-smoker subsample (14.4 sites, sd
< 0.05). Smokers had a mean
sd
1.6 respectively;
PD of 3.12 mm (sd
0.5) compared to 2.94 mm (sd
0.4) for the subsample of non-smokers (P < 0.05). Additional clinical periodontal characteristics of smokers (n
63) and the non-smoker subsample (n 126) are presented
in Table 1. Statistically significant differences between smokers and the non-smoker subsample in the percent of subjects
with at least one site having a PD > 3.5 mm, PD > 4.5 mm,
and PD > 5.5 mm were identified. Over 75% of the smokers
had at least one site with a PD > 3.5 mm compared to
approximately 60% of the non-smoker subsample. There were
no statistically significant differences between the percent of
smokers and the subsample of non-smokers with at least one
site of GI > 0, GI > 1, or bleeding on probing.
The number of smokers and subsample non-smokers
matched for age, sex, plaque, and calculus with a mean PD
=

1227

3.5

mm or <

subsample.

The results of univariate analyses for the association besmoking, selected bacteria, and periodontal disease
severity (mean PD > 3.5 mm vs. mean PD < 3.5 mm) are
presented in Table 3. Of the variables examined, 4 (smoking, A. actinomycetemcomitans, P. intermedia, and. corrodens) were significantly associated with disease severity
(P < 0.05). The odds ratio for P. gingivalis and F. nucleatum did not reach statistical significance in this model.
tween

DISCUSSION
In this study of primarily healthy, middle-aged adults, plaque
and calculus were correlated with mean probing depth. The
correlations were low indicating a weak relationship between these variables. However, the results are consistent
with previous studies which indicate that plaque46 and
calculus27 are associated with periodontal status.
Clinical periodontal indices and probing depth measurements of smokers and the randomly drawn subsample of
non-smokers matched for age, sex, plaque, and calculus
revealed that, on average, subjects had gingival inflammation and/or shallow pockets. More smokers had increased pocket depth (mean PD > 3.5 mm) than the subsample of non-smokers. An average of 14.9 sites per subject
were measured indicating that most subjects had all premolare and molars (excluding third molars) in the selected
sextant. Smokers had fewer sites measured however, indicating they had fewer teeth than the non-smoker subsample in the selected sextant. Less than 60% of smokers had
4 teeth in the randomly measured sextant. Previous studies
have reported a greater rate of tooth loss in smokers than
non-smokers.6 7'10"12 The results of this study lend support
to this finding.
In the present study, periodontal disease severity was
greater in smokers than the non-smoker subsample as evidenced by a greater percentage of subjects with at least one
PD > 3.5 mm, > 4.5 mm, and > 5.5 mm. Over 25% of
the smokers had at least one pocket of moderate depth (PD
> 4.5
mm) compared to approximately 10% of the nonsmoker subsample. In addition, 24% of the smokers had a
mean PD > 3.5 mm compared to only 6% of the nonsmoker subsample. These findings are consistent with an
earlier report which indicated an increase of probing depth
in smokers when compared to non-smokers.8
No significant differences existed between smokers and the

1228

CIGARETTE

SMOKING, BACTERIAL PATHOGENS, AND

J Periodontol
December 1993

PERIODONTAL STATUS

Table 1. Clinical Periodontal Characteristics of Cigarette Smokers and the

Matched for Age, Sex, Plaque, and Calculus

Subsample of Non-Smokers

Non-

Smoker

Subsample
Subjects
(n 126)

Smokers
% Subjects

(n

Characteristic
one site with:

Total

Subjects
(n 189)

63)

At least

Probing depth

>

3.5

mm

76.2

(48)

Probing depth

>

4.5

mm

25.4

(16)

Probing depth > 5.5 mm

Gingival index > 0
Gingival index > 1
Bleeding on probing > 0
Bleeding on probing > 1
*/<0.05; chi-square

tests

Depth

Smoking Status
Smokers (n
63)
Non-smoker subsample (n
=

15/48
7/119

126)

5.3; 95% Cl

<

2.0

3.5

mm

48
119

15
7

Odds Ratio

Depth

Mean Probing
3.5 mm

13.
-

subsample in the prevalence of P. gingivalis, A.

actinomycetemcomtitans, P. intermedia, E. corrodens or F.
nucleatum (P > 0.05). While the prevalence rates for these
bacteria (40 to 50%) are relatively high, they are lower than
some previous studies.38-47-48 These differences may be attributed differences in detection techniques, type of
disease,49-50 number and location of sites samples,51-52 and
non-smoker

oral health.
Of the variables examined as potential indicators, cigarette smoking demonstrated the strongest association with
periodontal disease severity. The odds of smokers having
Table 3. Univariate Model for the Association Between Smoking,
Selected Bacteria, and Mean Probing Depth (PD > 3.5 mm vs. PD
< 3.5 mm) for Smokers (n = 63) and the Non-Smoker
Subsample
(n = 126) Matched for Age, Sex, Plaque, and Calculus

Variable

Smoking

A. actinomycetemcomitans
P. intermedia
E. corrodens
P. gingivalis
F. nucleatum

Odds
Ratio

95% Confidence
Interval

5.31
2.73
2.75
2.90
1.90
1.25

2.0- 13.8*
1.1
6.7t
1.1
6.9t
1.1
7.8t
0.8- 4.7
0.5
3.1
-

<

<

0.001 for odds ratio

0.05.

being different

from 1.0.

I 12.7(08)
100.0
1.6
93.7
60.3

for each characteristic

Table 2. Number of Smokers and Subsample Non-Smokers,

Matched for Age, Sex, Plaque, and Calculus by Mean Probing

(63)
(01)
(59)
(38)

were

based

on

59.5

(75)

10.3

(13)

2.4
100.0
4.0
99.2
60.3

(03)
(126)
(05)
(125)
(76)

presence

or

65.1

(123)

15.3

(29)

5.8
100.0
3.2
97.4
60.3

(11)
(189)
(06)
(184)
(114)

absence of such sites in

subject.

a mean PD > 3.5 mm were 5.3 times greater than for the
non-smoker subsample. Smoking has been previously associated with a 5 fold greater risk of adult Periodontitis.23
The odds of smokers being among those receiving periodontal treatment have been reported to be 2 to 3 times
greater than of patients in the general practice from which
they were referred53 and the population at large.8 Additionally, patients with refractory Periodontitis showed a 4.5 fold
greater prevalence of smoking than the general population.13
The association between cigarette smoking and severity
of periodontal disease in this study is of particular interest
since subjects were medically healthy, had access to health
care, and, on average, had received recent medical and
dental examinations.34 The percent of subjects who reported
smoking was less than half that reported for adults in
Minnesota.34 In addition, variables commonly associated
with increased prevalence and severity of periodontal disease (plaque, calculus, age, and sex) were controlled. Prevalences of selected bacteria associated with periodontal disease were examined and found not to differ between smokers
and the non-smoker subsample. Therefore, several factors
which can contribute to periodontal disease susceptibility
were minimized in this sample. Yet, a strong association
remained between smoking and increased probing depth
indicating that smoking per se has an adverse affect on

periodontal

status.

The bacterial variables of A. actinomycetemcomitans, P.

intermedia, and E. corrodens also contributed information
regarding periodontal disease severity but to a lesser degree
than smoking. The odds of subjects with A. actinomycetemcomitans, P. intermedia, or E. corrodens having a mean
PD > 3.5 mm were 2 to 3 times greater than for subjects
without the bacterium. Since it is becoming clear that the
presence of bacterial pathogens alone is not sufficient for
disease activity to occur,54 interactions between bacterial
variables and smoking or other unknown factors may have

Volume 64
Number 12

STOLTENBERG, OSBORN, PIHLSTROM, HERZBERG, AEPPLI, WOLFF, FISCHER

contributed to these results. In this study, multivariate analyses necessary to examine these interactions were not possible due to the limited number of subjects with a mean PD
> 3.5 mm. Future
investigations which include these analyses would be of value.

CONCLUSIONS
The following conclusions are made from this study:
1. After matching for age, sex, plaque, and calculus, the
odds of having a mean posterior proximal probing depth >
3.5 mm were 5.3 times greater for smokers than non-smokers.
2. The prevalence of P. gingivalis, A. actinomycetemcomtitans, P. intermedia, E. corrodens, or F. nucleatum
did not differ between smokers and non-smokers.
3. A. actinomycetemcomitans, P. intermedia, E. corrodens, and smoking were each associated with increased

posterior proximal probing depth. However, cigarette

smoking was a stronger risk indicator for the presence of a
mean probing depth > 3.5 mm than any of 5 bacteria commonly associated with periodontal disease.
mean

Acknowledgments
The authors gratefully acknowledge the clinical assistance
of Beverly A. Huso and the statistical advice of Nancy A.
Hardie, University of Minnesota Clinical Research Center
for Periodontal Diseases. Supported by NIH-NIDR grants
P50-DE08489 and P30-DE09737.

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Send reprint requests to: Jill L. Stoltenberg, University of Minnesota,

School of Dentistry, Department of Preventive Sciences, 9-436 Moos HST,
515 Delaware Street S.E., Minneapolis, MN 55455.
Accepted for publication June 9, 1993.