Evaluation of the subgingival microbiota of alcoholic and non-alcoholic individuals

Evaluation of the subgingival microbiota of alcoholic and non-alcoholic individuals

Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 729-738, Copyright © 2011 Elsevier Ltd

Abstract

Objectives

The aim of this study was to evaluate the composition of the subgingival microbiota of alcoholic and non-alcoholic individuals.

Methods

The study was conducted with 49 alcoholic and 49 non-alcoholic males of the Philippe Pinel Institute, Rio de Janeiro, Brazil. The subjects were selected by convenience and two criteria were used to diagnose alcohol dependence: the CAGE (cut-down, annoyed, guilt, eyes-opener) questionnaire and the International Statistical Classification of Diseases, 10th edition (WHO). Subgingival biofilm samples were obtained from 4 sites, 2 with probing depth (PD) ≥ 4 mm and 2 sites with PD < 4 mm. The presence and levels of 45 bacterial taxa were analysed using the checkerboard technique.

Results

The prevalence of bacterial species was not significantly different between groups. However, alcoholics showed significantly higher mean counts of Aggregactibacter actinomycetemcomitans , Fusobacterium nucleatum nucleatum , and Porphyromonas gingivalis (adjusted p < 0.001). Moreover, alcoholics harboured significantly higher mean levels of Capnocytophaga sputigena , Fusobacterium nucleatum vincentii , F. nuc. nucleatum , Gemella morbillorum , Neisseria mucosa , P. gingivalis , Streptococcus gordonii , and Tannerella forsythia at sites with PD <4 mm or ≥4 mm compared to non-alcoholics ( p ≤ 0.001). Of interest, shallow sites of alcoholics presented significantly higher mean levels of F. nuc. vincentii , F. nuc nucleatum , P. gingivalis , and T. forsythia than sites with PD ≥ 4 mm of non-alcoholics ( p ≤ 0.001).

Conclusions

Alcoholics and non-alcoholics present a diverse and complex microbiota; however, alcoholics harbour significantly higher levels of periodontopathic species in the subgingival microbiota than non-alcoholics.

Introduction

Alcohol dependence is considered as one of the most serious medical problems. The incidence of excessive ingestion of alcohol is the third cause of death in the world, after cancers and cardiovascular complications. Alcohol consumption in the Brazilian population is estimated in 68%, and 11.2% out of those present alcohol dependence. There are direct toxic damages related to alcohol consumption such as fatty liver cirrhosis, cerebral atrophy, cardiomiopathy, gastrointestinal bleeding and pancreatitis. Alcohol dependence also causes indirect oral problems including caries, tooth loss, periodontal disease, and cancers. In addition, studies which investigated the effects of alcohol in the periodontium showed that alcoholics have more risk to develop periodontal diseases.

The severity of periodontal disease in alcohol abusers could be explained by the disturbed defence mechanisms, which includes impaired neutrophils, macrophage and T-cell functions, and increased frequency of infections. Furthermore, the toxic effects of alcohol abuse on liver can interfere with protein metabolism and tissue healing.

Periodontal disease is a polimicrobial infection of the periodontium caused by specific pathogenic bacteria that leads to chronic inflammation and periodontal tissue breakdown. There is enough evidence to consider the imbalance between the pathogenic periodontal subgingival microbiota and the host response as the main cause of destructive periodontal diseases. In addition, this host–parasite relationship is modulated by environmental, behavioural and genetic factors. Amongst these factors, alcohol consumption may have a significant impact on the homeostase between periodontal bacteria and host response. However, no study has examined the composition of the subgingival periodontal microbiota and its relationship with periodontal disease development in alcoholic individuals. Thus, the purpose of the present study was to describe the subgingival microbiota of alcoholic and non-alcoholic individuals by the Checkerboard DNA–DNA hybridization technique.

Materials and methods

Study population

The subject population and clinical assessment of this cross-sectional study has been previously described. Briefly, this sample was comprised of 49 non-alcoholic and 49 alcoholic male individuals. All subjects were informed about the nature of the study, and a signed consent form was obtained. The study protocol was approved by the Committee for Human Subjects at the Philippe Pinel Institute, Rio de Janeiro.

The alcoholic group was selected from a group of patients attending an Alcoholic Treatment Unit (UTA) at Philippe Pinel Institute, Rio de Janeiro, Brazil. Visitors to in-patients at the same hospital composed the non-alcoholic group. The alcoholic group were diagnosed as alcohol-dependent currently abstenic in accordance to the International Classification of Diseases, 10th Revision (ICD-10) criteria and the use of the questionnaire CAGE (cut-down, annoyed, guilt, eye-opener). Both groups were balanced for smoking. A questionnaire on socio-demographic data with questions about smoking habits, income, education, and living conditions was also applied.

Selected subjects were ≥35 years of age, had at least 15 teeth and had not been submitted to antibiotic medication or periodontal therapy in the previous 6 months. In addition, individuals who were cocaine users, who presented necrotizing ulcerative gingivitis or periodontitis, and systemic conditions associated with periodontal disease such as Diabetes Mellitus and AIDS were not included in this sample population.

Clinical examinations were performed by a single calibrated examiner. Periodontal full-mouth measurements including probing depth (PD), clinical attachment level (CAL), presence or absence of supragingival biofilm (VP) and bleeding on probing (BOP) were recorded at 6 sites per tooth, excluding third molars. Socio-demographics and clinical data are presented in Table 1 .

Table 1
Socio-demographic and clinical parameters of alcoholic and non-alcoholic subjects.
Parameter Alcoholic ( N = 49) Non-alcoholic ( N = 49) p Value
Age [median (range)] 46.0 (30.0) 42.0 (27.0) 0.006 *
Income [≤6 minimum wages/month; n (%)] 42 (85.7) 40 (81.6) 0.580
Education [≤8 years; n (%)] 31 (63.3) 23 (46.9) 0.100
Living alone [ n (%)] 12 (24.5) 12 (24.5) 1.000
Smokers § [yes; n (%)] 32 (65.3) 32 (65.3)
CAL [mean (SD)] 3.5 (1.7) 2.9 (1.2) 0.001 ||
PD [mean (SD)] 3.2 (1.2) 2.7 (1.0) <0.001 ||
VP [mean % of sites (SD)] 58.9 (31.6) 51.0 (2.52) 0.176 ||
BOP [mean % of sites (SD)] 19.2 (25.0) 13.9 (18.9) 0.240 ||
CAL: clinical attachment level; PD: probing depth; VP: supragingival biofilm; BOP: bleeding on probing.

* Mann–Whitney test.

Brazilian minimum wage = $223.53 (US dollar) per month (2007).

χ 2 test.

§ Groups were controlled for smoking.

|| t Test.

Microbiological assessment

Supragingival biofilm was removed with sterile gauze and individuals’ samples of subgingival biofilm were taken using sterile Gracey curettes. f

f Hu-Friedy, Chicago, IL, USA.

Two samples were obtained from sites with PD < 4 mm, and 2 samples from sites with PD ≥ 4 mm.

The presence and levels of 45 bacterial taxa ( Table 2 ) were determined in the biofilm samples by genomic DNA probes and the Checkerboard DNA–DNA hybridization method. In brief, the bacterial cells were lysed and denatured DNA fixed in individual lanes on a nylon membrane g

g Hu-Friedy, Chicago, IL, USA.

using a slot blot device h

h GE Healthcare Life Science, São Paulo, SP, Brazil.

. Forty-five digoxigenin-labelled i

i Minislot 30, Miniblotter 45, Immunetics, Cambridge, MA, USA.

whole genomic probes were hybridized perpendicularly to the lanes of the bacterial samples. Bound probes were detected using phosphatase-conjugated antibody to digoxigenin j

j Roche Applied Science, São Paulo, SP, Brazil.

and fluorescence captured by an imaging system. k

k Storm™ 860 imaging system, GE Healthcare Life Science.

Signals were evaluated visually by comparison with the standards at 10 5 and 10 6 bacterial cells for the test species on the same membrane. They were recorded as: 0, not detect; 1, <10 5 cells; 2, approximately 10 5 ; 3, 10 5 –10 6 ; 4, approximately 10 6 , and 5, >10 6 cells. Failure to detect a signal was recorded as zero, although counts in the 1–1000 ranges could have been present. The sensitivity and specificity of these probes were determined as reported by Socransky et al.
Table 2
Bacterial strains used for the construction of whole genomic DNA probes tested against subgingival biofilm samples.
Bacterial species Strains a Bacterial species Strains a
Acinetobacter baumannii 19606 Helicobacter pylori 43504
Aggregatibacter actinomycetemcomitans a 43718 Leptotrichia buccalis 14201
Aggregatibacter actinomycetemcomitans b 29523 Neisseria mucosa 19696
Actinomyces gerencseriae 23860 Parvimonas micra 33270
Actinomyces israelii 12102 Porphyromonas gingivalis 33277
Actinomyces odontolyticus 17929 Pseudomonas aeruginosa 27853
Actinomyces naeslundii I 12104 Prevotella intermedia 25611
Actinomyces oris 43146 Prevotella melaninogenica 25845
Capnocytophaga gingivalis 33624 Prevotella nigrescens 33563
Capnocytophaga ochraceae 33596 Propionibacterium acnes 11827
Campylobacter rectus 33238 Selenomonas noxia 43541
Capnocytophaga sputigena 33612 Staphylococcus aureus 33591
Campylobacter showae 51146 Streptococcus anginosus 33397
Eubacterium nodatum 33099 Streptococcus constellatus 27823
Eikenella corrodens 23834 Streptococcus gordonii 10558
Eubacterium saburreum 33271 Streptococcus intermedius 27335
Enterococcus faecalis 29212 Streptococcus oralis 35037
Escherichia coli 33780 Streptococcus mitis 49456
Fusobacterium nucleatum nucleatum 25586 Streptococcus sanguinis 10556
Fusobacterium periodonticum 33693 Tannerella forsythia 43037
Fusobacterium nucleatum polymorphum 10953 Treponema denticola B1 b
Fusobacterium nucleatum vincentii 49256 Veillonella parvula 10790
Gemella morbilorum 27824

a ATCC (American Type Culture Collection, Rockville, MD).

b The Forsyth Institute (Boston, MA).

Statistical analysis

All statistical tests were performed using a statistical software package. l

l Statistical Package for the Social Sciences, SPSS, release 17.0, Chicago, IL, USA.

Clinical parameters were analysed for each individual and then averaged across individuals within the two clinical groups. Intergroup comparisons of socio-demographic and periodontal clinical data were analysed by Mann–Whitney, χ 2 and t tests. Microbial data were expressed as frequency and mean levels of each species in the two groups. The levels (scores 0–5) of each species in a sample were converted to absolute numbers and log 10 transformed for presentation in graphics. Differences in the prevalence and levels of the species were determined by Chi-square and Mann–Whitney tests, respectively. Comparisons between the two groups for sites with PD < 4 mm and PD ≥ 4 mm were examined by the Mann–Whitney test, considering the subject the unit of analysis. Adjustments for multiple comparisons of microbiological data were made as described by Socransky et al. In brief, a significance level of 0.05 for all 45 comparisons (45 bacterial species) requires a p ≤ 0.001 for each individual comparison to be considered statistically significant where 0.05 = 1 − (1 − p ) 45 .

Results

The prevalence of the tested bacteria is presented in Fig. 1 . Species detected in all samples of subjects in both groups were Leptotrichia buccalis, Prevotella nigrescens, Streptococcus constellatus, Streptococcus anginosus, Streptococcus gordonii, Streptococcus sanguinis, Selenomonas noxia , and Staphylococcus aureus. In the alcoholic group, Fusobacterium nuleatum nucleatum, Gemella morbillorum, Neisseria mucosa, Propionibacterium acnes, Porphyromonas gingivalis, Prevotella melaninogenica , and Streptococcus intermedius were present in all subgingival samples, whereas in the non-alcoholic group only the species Parvimonas micra and Streptococcus oralis present in a prevalence of 100%. Prevalence values did not differed significantly between groups at adjusted p ≤ 0.001 for any species.

Stacked bar chart of frequency (%) of detection of 45 bacterial taxa in subgingival biofilm samples from 49 non-alcoholic and 49 alcoholic subjects. Aa : Aggregatibacter actinomycetemcomitans .
Fig. 1
Stacked bar chart of frequency (%) of detection of 45 bacterial taxa in subgingival biofilm samples from 49 non-alcoholic and 49 alcoholic subjects. Aa : Aggregatibacter actinomycetemcomitans .

Regarding bacterial mean counts, only Actinomyces oris and Eubacterium saburreum were detected in low levels (<10 4 bacterial cells or log 4) in alcoholic and non-alcoholic subjects, respectively. Overall, alcoholics showed significantly higher mean bacterial counts of Aggregactibacter actinomycetemcomitans a, F. nucleatum nucleatum, and P. gingivalis ( p < 0.001) than non-alcoholics. The remaining species did not differ significantly between groups ( Fig. 2 ).

Stacked bar chart of bacterial mean counts (in log 10) of 45 oral taxa in subgingival biofilm samples from 49 non-alcoholic and 49 alcoholic subjects. Aa : Aggregatibacter actinomycetemcomitans. *Significant differences at adjusted p ≤ 0.001, Mann–Whitney test.
Fig. 2
Stacked bar chart of bacterial mean counts (in log 10) of 45 oral taxa in subgingival biofilm samples from 49 non-alcoholic and 49 alcoholic subjects. Aa : Aggregatibacter actinomycetemcomitans. *Significant differences at adjusted p ≤ 0.001, Mann–Whitney test.

Fig. 3 shows the mean bacterial counts in samples from sites with shallow PD (<4 mm) in both groups. Comparisons between groups for shallow sites demonstrated that alcoholic subjects harboured significantly higher mean counts of S. gordonii, Capnocytophaga sputigena, F. nucleatum vincentii, F. nucleatum nucleatum, P. gingivalis, Tannerella forsythia, G. morbillorum, and N. mucosa, and lower levels of S. anginosus and Pseudomonas aeruginosa compared to non-alcoholic subjects ( p < 0.001).

Stacked bar chart of bacterial mean counts (in log 10) of 45 oral taxa in subgingival biofilm samples obtained from sites with probing depth (PD) <4 mm of 49 non-alcoholic and 49 alcoholic subjects. The species were ordered according to the complexes described by Socransky et al. 27 Aa : Aggregatibacter actinomycetemcomitans. *Significant differences at adjusted p ≤ 0.001 between groups; Mann–Whitney test.
Fig. 3
Stacked bar chart of bacterial mean counts (in log 10) of 45 oral taxa in subgingival biofilm samples obtained from sites with probing depth (PD) <4 mm of 49 non-alcoholic and 49 alcoholic subjects. The species were ordered according to the complexes described by Socransky et al. Aa : Aggregatibacter actinomycetemcomitans. *Significant differences at adjusted p ≤ 0.001 between groups; Mann–Whitney test.

In sites with PD ≥ 4 mm, alcoholics showed significant higher levels of S. gordonii, C. sputigena, F. nucleatum vincentii, F. nucleatum nucleatum, P. gingivalis, T. forsythia, G. morbillorum, N. mucosa , and S. anginosus ( p ≤ 0.001) than non-alcoholics ( Fig. 4 ). Surprisingly, when comparisons were performed between shallow sites from alcoholics and sites with PD ≥ 4 mm from non-alcoholics, alcoholics had significantly higher mean counts of S. gordonii, C. sputigena, F. nucleatum vicentii, F. nucleatum nucleatum, P. gingivalis, T. forsythia, G. morbillorum, N. mucosa , and S. anginosus ( p ≤ 0.001) than non-alcoholics ( Figs. 3 and 4 ).

Stacked bar chart of bacterial mean counts (in log 10) of 45 oral taxa in subgingival biofilm samples obtained from sites with probing depth (PD) ≥4 mm of 49 non-alcoholic and 49 alcoholic subjects. The species were ordered according to the complexes described by Socransky et al. 27 Aa : Aggregatibacter actinomycetemcomitans. *Significant differences at adjusted p ≤ 0.001 between groups; Mann–Whitney test.
Fig. 4
Stacked bar chart of bacterial mean counts (in log 10) of 45 oral taxa in subgingival biofilm samples obtained from sites with probing depth (PD) ≥4 mm of 49 non-alcoholic and 49 alcoholic subjects. The species were ordered according to the complexes described by Socransky et al. Aa : Aggregatibacter actinomycetemcomitans. *Significant differences at adjusted p ≤ 0.001 between groups; Mann–Whitney test.

Discussion

Alcoholism and alcohol abuse is related to harmful effects on host due the collateral effects and the long term presence of alcohol on the body. Alcoholism leads to damage on systemic health, and it is a risk factor for oral infections as well. The direct effects of alcohol on oral cavity are related with oropharynx cancers, caries and periodontal diseases. Alcohol has also a special property to change the oral microenvironment due to its anti-microbial effect against periodontal pathogens such as A. actinomycetemcomitans and P. gingivalis. In the current study, we evaluated the composition of the subgingival microbiota of subjects with a diagnostic of alcohol-dependent currently abstenic in comparison to non-alcoholic individuals. Amongst the available studies, Tezal et al. evaluated the presence of eight bacteria using immunofluorescence technique in subgingival biofilm samples from alcohol consumers. The authors showed that high-drinkers harboured slightly higher frequencies of T. forsythia and P. gingivalis. In another investigation using real-time polymerase chain reaction, the levels of S. anginosus in the saliva of alcoholics were higher than that in periodontitis patients, gastritis patients, esophageal cancer patients and healthy people. Yokoyama et al. studied alcoholics subjects and observed that salivary yeast and total microorganism counts were significantly and independently correlated with salivary acetaldehyde production. Furthermore, after three weeks of abstinence, the levels of α-hemolytic streptococci, yeasts and all microorganisms were significantly reduced. These studies usually evaluate only the presence of a reduced number of samples and bacterial species. Considering the complexity and variability of the periodontal microbiota within and amongst subjects, particularly in the levels of periodontal pathogens, the use of methods that do not quantify species or are limited to a small range of microorganisms and samples may lead to incomplete or conflicting results. To overcome these limitations, we employed the checkerboard DNA–DNA hybridization technique. This method provided us with a wider view of the predominant microbial profiles of individuals in both clinical groups.

Regarding the prevalence of the species evaluated, we did not found significant differences between groups. It is expected that the non-alcoholic individuals present a more balanced oral environment, composed of higher proportions of beneficial species and lower prevalence of pathogens. Moreover, these subjects seem to have a good ecologic diversity, which compensates the high prevalence of some putative pathogens. From this ecologic point of view, the simple presence of periodontal pathogens in the biofilm does not necessarily determine the presence of or risk for destructive periodontal disease. In fact, the presence of oral pathogens in the subgingival microbiota of healthy subjects is not an unusual finding. However, the lack of difference in the prevalence of species between groups may due to the limited number of samples analysed, which probably does not represent the whole subgingival environment.

On the other hand, the impact of alcohol exposure on the levels of species in the subgingival microbiota was evident. For instance, three putative periodontal pathogens ( F. nucleatum nucleatum, P gingivalis , and A. actinomycetemcomitans ) were detected in higher mean counts in subgingival samples from alcoholics compared to non-alcoholics. These bacteria are strongly related to the evolution of chronic periodontal disease and strongly associated to deep PD and sites with bleeding on probing. Moreover, P gingivalis and A. actinomycetemcomitans are recognized as classic periodontal pathogens involved in the etiopathogenesis of chronic and aggressive periodontitis. These microbiological findings are in accordance with the clinical characteristics of this study population reported previously by Amaral et al. The authors demonstrated that the alcoholic subjects had significantly higher mean CAL and PD, as well higher mean proportion of sites with CAL and PD ≥ 4 mm compared to non-alcoholic subjects.

An intriguing result observed in this study was the significantly higher levels of members of the orange ( F. nucleatum vincentii, F. nucleatum nucleatum ) and red ( P. gingivalis and T. forsythia ) complexes in samples from shallow sites (PD < 4 mm) of alcoholics compared to shallow sites or sites with PD ≥ 4 mm in non-alcoholics. These data may indicate that long term exposure to alcohol has a greater impact on shallow sites than sites where marked local environmental changes (periodontal pockets) have already occurred. A similar finding was reported by Haffajee and Socransky regarding the impact of smoking on the subgingival microbiota. A significantly higher prevalence of species of the orange and red complexes was observed in shallow sites of smokers compared to non-smokers; however, no differences were found for sites with deep pockets. In contrast, other authors showed that sites with periodontal pockets of individuals with an IL-1 genotype positive presented significantly higher counts of these pathogens than periodontal pockets of genotype negative subjects. When shallow sites were evaluated, no differences in the proportions of these species between different IL-1 polymorphisms were detected. Thus, genetic and/or environmental factors may have different effects on the composition of the subgingival microbiota of sites with distinct periodontal clinical features. Another explanation for our findings would be the fact that these alcoholic individuals presented higher proportions of sites with periodontal pocket and inflammation. Some studies have reported that healthy sites (shallow PD) of individuals with periodontitis harbour higher prevalence and levels of periodontal pathogens compared to sites in periodontally healthy individuals. Whether high levels of pathogenic bacteria in healthy sites of alcoholics will increase the risk of those sites to present future periodontal attachment loss needs further investigation. Nevertheless, the deleterious effect of alcohol on the balance between this microbiota and the host immune system will probably contribute to this event.

Species of medical importance, not usually considered as periodontal pathogens were also detected in the subgingival microbiota of individuals in both groups. Only P. aeruginosa was found in a significantly higher level in shallow sites of non-alcoholics compared to alcoholics. Similarly, Gonçalves et al. reported a higher prevalence of P. aeruginosa in the shallow sites of HIV-infected than non-infected subjects. Thus, one could argue that immunosuppression either by HIV infection or alcohol consumption may favour colonization by this non-oral pathogen. Other studies have also shown the presence of P. aeruginosa in sites with deep periodontal pockets. P. aeruginosa is a pathogen associated with major medical diseases such as respiratory and urinary tract infections, as well as nosocomial bloodstream infections. Therefore, close attention should be given to his pathogen in the oral microbiota, especially in individuals at greater risk for infections such as individuals with immunological impairments. Furthermore, studies evaluating the impact of high levels of periodontopathogens in shallow sites of alcoholic subjects are needed to clarify the etiopathogenesis of periodontitis and to provide more adequate preventive and therapeutic approaches for these subjects.

Conclusions

In summary, the present investigation showed that the subgingival microbiota of alcoholic and non-alcoholic subjects is diverse and complex. Species of the orange and red complexes are detected in significantly high levels in subgingival samples of alcoholics, even in sites with shallow probing depths.

Acknowledgments

This study was supported in part by National Council for Scientific and Technological Development (CNPq) , Coordination of Improvement of Higher Education Personnel (CAPES), Brasilia, Brazil ; and Foundation for Research Financial Support in the State of Rio de Janeiro (FAPERJ), Rio de Janeiro, Brazil . The authors declare that they do not have any conflict of interest regarding the present study.

d Address: Universidade Federal do Rio de Janeiro – Faculdade de Odontologia, Departamento de Clínica Odontológica – Disciplina de Periodontia, Avenida Brigadeiro Trompovsky S/N – Bloco “K”, Cidade Universitária, Rio de Janeiro – Brazil – CEP: 21941-590.

e Address: Universidade Federal do Rio de Janeiro – Instituto de Microbiologia Professor Paula de, Góes, Av. Carlos Chagas Filho, 373. Edifício do Centro de Ciências da Saúde, Bloco “I”, Cidade Universitária, Rio de Janeiro – Brazil – CEP: 21941-902.

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Evaluation of the subgingival microbiota of alcoholic and non-alcoholic individuals Cristine da Silva Furtado Amaral , Carina Maciel da Silva-Boghossian , Anna Thereza Thomé Leão and Ana Paula Vieira Colombo Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 729-738, Copyright © 2011 Elsevier Ltd Abstract Objectives The aim of this study was to evaluate the composition of the subgingival microbiota of alcoholic and non-alcoholic individuals. Methods The study was conducted with 49 alcoholic and 49 non-alcoholic males of the Philippe Pinel Institute, Rio de Janeiro, Brazil. The subjects were selected by convenience and two criteria were used to diagnose alcohol dependence: the CAGE (cut-down, annoyed, guilt, eyes-opener) questionnaire and the International Statistical Classification of Diseases, 10th edition (WHO). Subgingival biofilm samples were obtained from 4 sites, 2 with probing depth (PD) ≥ 4 mm and 2 sites with PD < 4 mm. The presence and levels of 45 bacterial taxa were analysed using the checkerboard technique. Results The prevalence of bacterial species was not significantly different between groups. However, alcoholics showed significantly higher mean counts of Aggregactibacter actinomycetemcomitans , Fusobacterium nucleatum nucleatum , and Porphyromonas gingivalis (adjusted p < 0.001). Moreover, alcoholics harboured significantly higher mean levels of Capnocytophaga sputigena , Fusobacterium nucleatum vincentii , F. nuc. nucleatum , Gemella morbillorum , Neisseria mucosa , P. gingivalis , Streptococcus gordonii , and Tannerella forsythia at sites with PD <4 mm or ≥4 mm compared to non-alcoholics ( p ≤ 0.001). Of interest, shallow sites of alcoholics presented significantly higher mean levels of F. nuc. vincentii , F. nuc nucleatum , P. gingivalis , and T. forsythia than sites with PD ≥ 4 mm of non-alcoholics ( p ≤ 0.001). Conclusions Alcoholics and non-alcoholics present a diverse and complex microbiota; however, alcoholics harbour significantly higher levels of periodontopathic species in the subgingival microbiota than non-alcoholics. 1 Introduction Alcohol dependence is considered as one of the most serious medical problems. The incidence of excessive ingestion of alcohol is the third cause of death in the world, after cancers and cardiovascular complications. Alcohol consumption in the Brazilian population is estimated in 68%, and 11.2% out of those present alcohol dependence. There are direct toxic damages related to alcohol consumption such as fatty liver cirrhosis, cerebral atrophy, cardiomiopathy, gastrointestinal bleeding and pancreatitis. Alcohol dependence also causes indirect oral problems including caries, tooth loss, periodontal disease, and cancers. In addition, studies which investigated the effects of alcohol in the periodontium showed that alcoholics have more risk to develop periodontal diseases. The severity of periodontal disease in alcohol abusers could be explained by the disturbed defence mechanisms, which includes impaired neutrophils, macrophage and T-cell functions, and increased frequency of infections. Furthermore, the toxic effects of alcohol abuse on liver can interfere with protein metabolism and tissue healing. Periodontal disease is a polimicrobial infection of the periodontium caused by specific pathogenic bacteria that leads to chronic inflammation and periodontal tissue breakdown. There is enough evidence to consider the imbalance between the pathogenic periodontal subgingival microbiota and the host response as the main cause of destructive periodontal diseases. In addition, this host–parasite relationship is modulated by environmental, behavioural and genetic factors. Amongst these factors, alcohol consumption may have a significant impact on the homeostase between periodontal bacteria and host response. However, no study has examined the composition of the subgingival periodontal microbiota and its relationship with periodontal disease development in alcoholic individuals. Thus, the purpose of the present study was to describe the subgingival microbiota of alcoholic and non-alcoholic individuals by the Checkerboard DNA–DNA hybridization technique. 2 Materials and methods 2.1 Study population The subject population and clinical assessment of this cross-sectional study has been previously described. Briefly, this sample was comprised of 49 non-alcoholic and 49 alcoholic male individuals. All subjects were informed about the nature of the study, and a signed consent form was obtained. The study protocol was approved by the Committee for Human Subjects at the Philippe Pinel Institute, Rio de Janeiro. The alcoholic group was selected from a group of patients attending an Alcoholic Treatment Unit (UTA) at Philippe Pinel Institute, Rio de Janeiro, Brazil. Visitors to in-patients at the same hospital composed the non-alcoholic group. The alcoholic group were diagnosed as alcohol-dependent currently abstenic in accordance to the International Classification of Diseases, 10th Revision (ICD-10) criteria and the use of the questionnaire CAGE (cut-down, annoyed, guilt, eye-opener). Both groups were balanced for smoking. A questionnaire on socio-demographic data with questions about smoking habits, income, education, and living conditions was also applied. Selected subjects were ≥35 years of age, had at least 15 teeth and had not been submitted to antibiotic medication or periodontal therapy in the previous 6 months. In addition, individuals who were cocaine users, who presented necrotizing ulcerative gingivitis or periodontitis, and systemic conditions associated with periodontal disease such as Diabetes Mellitus and AIDS were not included in this sample population. Clinical examinations were performed by a single calibrated examiner. Periodontal full-mouth measurements including probing depth (PD), clinical attachment level (CAL), presence or absence of supragingival biofilm (VP) and bleeding on probing (BOP) were recorded at 6 sites per tooth, excluding third molars. Socio-demographics and clinical data are presented in Table 1 . Table 1 Socio-demographic and clinical parameters of alcoholic and non-alcoholic subjects. Parameter Alcoholic ( N = 49) Non-alcoholic ( N = 49) p Value Age [median (range)] 46.0 (30.0) 42.0 (27.0) 0.006 * Income † [≤6 minimum wages/month; n (%)] 42 (85.7) 40 (81.6) 0.580 ‡ Education [≤8 years; n (%)] 31 (63.3) 23 (46.9) 0.100 ‡ Living alone [ n (%)] 12 (24.5) 12 (24.5) 1.000 ‡ Smokers § [yes; n (%)] 32 (65.3) 32 (65.3) CAL [mean (SD)] 3.5 (1.7) 2.9 (1.2) 0.001 || PD [mean (SD)] 3.2 (1.2) 2.7 (1.0) <0.001 || VP [mean % of sites (SD)] 58.9 (31.6) 51.0 (2.52) 0.176 || BOP [mean % of sites (SD)] 19.2 (25.0) 13.9 (18.9) 0.240 || CAL: clinical attachment level; PD: probing depth; VP: supragingival biofilm; BOP: bleeding on probing. * Mann–Whitney test. † Brazilian minimum wage = $223.53 (US dollar) per month (2007). ‡ χ 2 test. § Groups were controlled for smoking. || t Test. 2.2 Microbiological assessment Supragingival biofilm was removed with sterile gauze and individuals’ samples of subgingival biofilm were taken using sterile Gracey curettes. f f Hu-Friedy, Chicago, IL, USA. Two samples were obtained from sites with PD < 4 mm, and 2 samples from sites with PD ≥ 4 mm. The presence and levels of 45 bacterial taxa ( Table 2 ) were determined in the biofilm samples by genomic DNA probes and the Checkerboard DNA–DNA hybridization method. In brief, the bacterial cells were lysed and denatured DNA fixed in individual lanes on a nylon membrane g g Hu-Friedy, Chicago, IL, USA. using a slot blot device h h GE Healthcare Life Science, São Paulo, SP, Brazil. . Forty-five digoxigenin-labelled i i Minislot 30, Miniblotter 45, Immunetics, Cambridge, MA, USA. whole genomic probes were hybridized perpendicularly to the lanes of the bacterial samples. Bound probes were detected using phosphatase-conjugated antibody to digoxigenin j j Roche Applied Science, São Paulo, SP, Brazil. and fluorescence captured by an imaging system. k k Storm™ 860 imaging system, GE Healthcare Life Science. Signals were evaluated visually by comparison with the standards at 10 5 and 10 6 bacterial cells for the test species on the same membrane. They were recorded as: 0, not detect; 1, <10 5 cells; 2, approximately 10 5 ; 3, 10 5 –10 6 ; 4, approximately 10 6 , and 5, >10 6 cells. Failure to detect a signal was recorded as zero, although counts in the 1–1000 ranges could have been present. The sensitivity and specificity of these probes were determined as reported by Socransky et al. Table 2 Bacterial strains used for the construction of whole genomic DNA probes tested against subgingival biofilm samples. Bacterial species Strains a Bacterial species Strains a Acinetobacter baumannii 19606 Helicobacter pylori 43504 Aggregatibacter actinomycetemcomitans a 43718 Leptotrichia buccalis 14201 Aggregatibacter actinomycetemcomitans b 29523 Neisseria mucosa 19696 Actinomyces gerencseriae 23860 Parvimonas micra 33270 Actinomyces israelii 12102 Porphyromonas gingivalis 33277 Actinomyces odontolyticus 17929 Pseudomonas aeruginosa 27853 Actinomyces naeslundii I 12104 Prevotella intermedia 25611 Actinomyces oris 43146 Prevotella melaninogenica 25845 Capnocytophaga gingivalis 33624 Prevotella nigrescens 33563 Capnocytophaga ochraceae 33596 Propionibacterium acnes 11827 Campylobacter rectus 33238 Selenomonas noxia 43541 Capnocytophaga sputigena 33612 Staphylococcus aureus 33591 Campylobacter showae 51146 Streptococcus anginosus 33397 Eubacterium nodatum 33099 Streptococcus constellatus 27823 Eikenella corrodens 23834 Streptococcus gordonii 10558 Eubacterium saburreum 33271 Streptococcus intermedius 27335 Enterococcus faecalis 29212 Streptococcus oralis 35037 Escherichia coli 33780 Streptococcus mitis 49456 Fusobacterium nucleatum nucleatum 25586 Streptococcus sanguinis 10556 Fusobacterium periodonticum 33693 Tannerella forsythia 43037 Fusobacterium nucleatum polymorphum 10953 Treponema denticola B1 b Fusobacterium nucleatum vincentii 49256 Veillonella parvula 10790 Gemella morbilorum 27824 a ATCC (American Type Culture Collection, Rockville, MD). b The Forsyth Institute (Boston, MA). 2.3 Statistical analysis All statistical tests were performed using a statistical software package. l l Statistical Package for the Social Sciences, SPSS, release 17.0, Chicago, IL, USA. Clinical parameters were analysed for each individual and then averaged across individuals within the two clinical groups. Intergroup comparisons of socio-demographic and periodontal clinical data were analysed by Mann–Whitney, χ 2 and t tests. Microbial data were expressed as frequency and mean levels of each species in the two groups. The levels (scores 0–5) of each species in a sample were converted to absolute numbers and log 10 transformed for presentation in graphics. Differences in the prevalence and levels of the species were determined by Chi-square and Mann–Whitney tests, respectively. Comparisons between the two groups for sites with PD < 4 mm and PD ≥ 4 mm were examined by the Mann–Whitney test, considering the subject the unit of analysis. Adjustments for multiple comparisons of microbiological data were made as described by Socransky et al. In brief, a significance level of 0.05 for all 45 comparisons (45 bacterial species) requires a p ≤ 0.001 for each individual comparison to be considered statistically significant where 0.05 = 1 − (1 − p ) 45 . 3 Results The prevalence of the tested bacteria is presented in Fig. 1 . Species detected in all samples of subjects in both groups were Leptotrichia buccalis, Prevotella nigrescens, Streptococcus constellatus, Streptococcus anginosus, Streptococcus gordonii, Streptococcus sanguinis, Selenomonas noxia , and Staphylococcus aureus. In the alcoholic group, Fusobacterium nuleatum nucleatum, Gemella morbillorum, Neisseria mucosa, Propionibacterium acnes, Porphyromonas gingivalis, Prevotella melaninogenica , and Streptococcus intermedius were present in all subgingival samples, whereas in the non-alcoholic group only the species Parvimonas micra and Streptococcus oralis present in a prevalence of 100%. Prevalence values did not differed significantly between groups at adjusted p ≤ 0.001 for any species. Fig. 1 Stacked bar chart of frequency (%) of detection of 45 bacterial taxa in subgingival biofilm samples from 49 non-alcoholic and 49 alcoholic subjects. Aa : Aggregatibacter actinomycetemcomitans . Regarding bacterial mean counts, only Actinomyces oris and Eubacterium saburreum were detected in low levels (<10 4 bacterial cells or log 4) in alcoholic and non-alcoholic subjects, respectively. Overall, alcoholics showed significantly higher mean bacterial counts of Aggregactibacter actinomycetemcomitans a, F. nucleatum nucleatum, and P. gingivalis ( p < 0.001) than non-alcoholics. The remaining species did not differ significantly between groups ( Fig. 2 ). Fig. 2 Stacked bar chart of bacterial mean counts (in log 10) of 45 oral taxa in subgingival biofilm samples from 49 non-alcoholic and 49 alcoholic subjects. Aa : Aggregatibacter actinomycetemcomitans. *Significant differences at adjusted p ≤ 0.001, Mann–Whitney test. Fig. 3 shows the mean bacterial counts in samples from sites with shallow PD (<4 mm) in both groups. Comparisons between groups for shallow sites demonstrated that alcoholic subjects harboured significantly higher mean counts of S. gordonii, Capnocytophaga sputigena, F. nucleatum vincentii, F. nucleatum nucleatum, P. gingivalis, Tannerella forsythia, G. morbillorum, and N. mucosa, and lower levels of S. anginosus and Pseudomonas aeruginosa compared to non-alcoholic subjects ( p < 0.001). Fig. 3 Stacked bar chart of bacterial mean counts (in log 10) of 45 oral taxa in subgingival biofilm samples obtained from sites with probing depth (PD) <4 mm of 49 non-alcoholic and 49 alcoholic subjects. The species were ordered according to the complexes described by Socransky et al. Aa : Aggregatibacter actinomycetemcomitans. *Significant differences at adjusted p ≤ 0.001 between groups; Mann–Whitney test. In sites with PD ≥ 4 mm, alcoholics showed significant higher levels of S. gordonii, C. sputigena, F. nucleatum vincentii, F. nucleatum nucleatum, P. gingivalis, T. forsythia, G. morbillorum, N. mucosa , and S. anginosus ( p ≤ 0.001) than non-alcoholics ( Fig. 4 ). Surprisingly, when comparisons were performed between shallow sites from alcoholics and sites with PD ≥ 4 mm from non-alcoholics, alcoholics had significantly higher mean counts of S. gordonii, C. sputigena, F. nucleatum vicentii, F. nucleatum nucleatum, P. gingivalis, T. forsythia, G. morbillorum, N. mucosa , and S. anginosus ( p ≤ 0.001) than non-alcoholics ( Figs. 3 and 4 ). Fig. 4 Stacked bar chart of bacterial mean counts (in log 10) of 45 oral taxa in subgingival biofilm samples obtained from sites with probing depth (PD) ≥4 mm of 49 non-alcoholic and 49 alcoholic subjects. The species were ordered according to the complexes described by Socransky et al. Aa : Aggregatibacter actinomycetemcomitans. *Significant differences at adjusted p ≤ 0.001 between groups; Mann–Whitney test. 4 Discussion Alcoholism and alcohol abuse is related to harmful effects on host due the collateral effects and the long term presence of alcohol on the body. Alcoholism leads to damage on systemic health, and it is a risk factor for oral infections as well. The direct effects of alcohol on oral cavity are related with oropharynx cancers, caries and periodontal diseases. Alcohol has also a special property to change the oral microenvironment due to its anti-microbial effect against periodontal pathogens such as A. actinomycetemcomitans and P. gingivalis. In the current study, we evaluated the composition of the subgingival microbiota of subjects with a diagnostic of alcohol-dependent currently abstenic in comparison to non-alcoholic individuals. Amongst the available studies, Tezal et al. evaluated the presence of eight bacteria using immunofluorescence technique in subgingival biofilm samples from alcohol consumers. The authors showed that high-drinkers harboured slightly higher frequencies of T. forsythia and P. gingivalis. In another investigation using real-time polymerase chain reaction, the levels of S. anginosus in the saliva of alcoholics were higher than that in periodontitis patients, gastritis patients, esophageal cancer patients and healthy people. Yokoyama et al. studied alcoholics subjects and observed that salivary yeast and total microorganism counts were significantly and independently correlated with salivary acetaldehyde production. Furthermore, after three weeks of abstinence, the levels of α-hemolytic streptococci, yeasts and all microorganisms were significantly reduced. These studies usually evaluate only the presence of a reduced number of samples and bacterial species. Considering the complexity and variability of the periodontal microbiota within and amongst subjects, particularly in the levels of periodontal pathogens, the use of methods that do not quantify species or are limited to a small range of microorganisms and samples may lead to incomplete or conflicting results. To overcome these limitations, we employed the checkerboard DNA–DNA hybridization technique. This method provided us with a wider view of the predominant microbial profiles of individuals in both clinical groups. Regarding the prevalence of the species evaluated, we did not found significant differences between groups. It is expected that the non-alcoholic individuals present a more balanced oral environment, composed of higher proportions of beneficial species and lower prevalence of pathogens. Moreover, these subjects seem to have a good ecologic diversity, which compensates the high prevalence of some putative pathogens. From this ecologic point of view, the simple presence of periodontal pathogens in the biofilm does not necessarily determine the presence of or risk for destructive periodontal disease. In fact, the presence of oral pathogens in the subgingival microbiota of healthy subjects is not an unusual finding. However, the lack of difference in the prevalence of species between groups may due to the limited number of samples analysed, which probably does not represent the whole subgingival environment. On the other hand, the impact of alcohol exposure on the levels of species in the subgingival microbiota was evident. For instance, three putative periodontal pathogens ( F. nucleatum nucleatum, P gingivalis , and A. actinomycetemcomitans ) were detected in higher mean counts in subgingival samples from alcoholics compared to non-alcoholics. These bacteria are strongly related to the evolution of chronic periodontal disease and strongly associated to deep PD and sites with bleeding on probing. Moreover, P gingivalis and A. actinomycetemcomitans are recognized as classic periodontal pathogens involved in the etiopathogenesis of chronic and aggressive periodontitis. These microbiological findings are in accordance with the clinical characteristics of this study population reported previously by Amaral et al. The authors demonstrated that the alcoholic subjects had significantly higher mean CAL and PD, as well higher mean proportion of sites with CAL and PD ≥ 4 mm compared to non-alcoholic subjects. An intriguing result observed in this study was the significantly higher levels of members of the orange ( F. nucleatum vincentii, F. nucleatum nucleatum ) and red ( P. gingivalis and T. forsythia ) complexes in samples from shallow sites (PD < 4 mm) of alcoholics compared to shallow sites or sites with PD ≥ 4 mm in non-alcoholics. These data may indicate that long term exposure to alcohol has a greater impact on shallow sites than sites where marked local environmental changes (periodontal pockets) have already occurred. A similar finding was reported by Haffajee and Socransky regarding the impact of smoking on the subgingival microbiota. A significantly higher prevalence of species of the orange and red complexes was observed in shallow sites of smokers compared to non-smokers; however, no differences were found for sites with deep pockets. In contrast, other authors showed that sites with periodontal pockets of individuals with an IL-1 genotype positive presented significantly higher counts of these pathogens than periodontal pockets of genotype negative subjects. When shallow sites were evaluated, no differences in the proportions of these species between different IL-1 polymorphisms were detected. Thus, genetic and/or environmental factors may have different effects on the composition of the subgingival microbiota of sites with distinct periodontal clinical features. Another explanation for our findings would be the fact that these alcoholic individuals presented higher proportions of sites with periodontal pocket and inflammation. Some studies have reported that healthy sites (shallow PD) of individuals with periodontitis harbour higher prevalence and levels of periodontal pathogens compared to sites in periodontally healthy individuals. Whether high levels of pathogenic bacteria in healthy sites of alcoholics will increase the risk of those sites to present future periodontal attachment loss needs further investigation. Nevertheless, the deleterious effect of alcohol on the balance between this microbiota and the host immune system will probably contribute to this event. Species of medical importance, not usually considered as periodontal pathogens were also detected in the subgingival microbiota of individuals in both groups. Only P. aeruginosa was found in a significantly higher level in shallow sites of non-alcoholics compared to alcoholics. Similarly, Gonçalves et al. reported a higher prevalence of P. aeruginosa in the shallow sites of HIV-infected than non-infected subjects. Thus, one could argue that immunosuppression either by HIV infection or alcohol consumption may favour colonization by this non-oral pathogen. Other studies have also shown the presence of P. aeruginosa in sites with deep periodontal pockets. P. aeruginosa is a pathogen associated with major medical diseases such as respiratory and urinary tract infections, as well as nosocomial bloodstream infections. Therefore, close attention should be given to his pathogen in the oral microbiota, especially in individuals at greater risk for infections such as individuals with immunological impairments. Furthermore, studies evaluating the impact of high levels of periodontopathogens in shallow sites of alcoholic subjects are needed to clarify the etiopathogenesis of periodontitis and to provide more adequate preventive and therapeutic approaches for these subjects. 5 Conclusions In summary, the present investigation showed that the subgingival microbiota of alcoholic and non-alcoholic subjects is diverse and complex. Species of the orange and red complexes are detected in significantly high levels in subgingival samples of alcoholics, even in sites with shallow probing depths. Acknowledgments This study was supported in part by National Council for Scientific and Technological Development (CNPq) , Coordination of Improvement of Higher Education Personnel (CAPES), Brasilia, Brazil ; and Foundation for Research Financial Support in the State of Rio de Janeiro (FAPERJ), Rio de Janeiro, Brazil . The authors declare that they do not have any conflict of interest regarding the present study. d Address: Universidade Federal do Rio de Janeiro – Faculdade de Odontologia, Departamento de Clínica Odontológica – Disciplina de Periodontia, Avenida Brigadeiro Trompovsky S/N – Bloco "K", Cidade Universitária, Rio de Janeiro – Brazil – CEP: 21941-590. e Address: Universidade Federal do Rio de Janeiro – Instituto de Microbiologia Professor Paula de, Góes, Av. Carlos Chagas Filho, 373. Edifício do Centro de Ciências da Saúde, Bloco "I", Cidade Universitária, Rio de Janeiro – Brazil – CEP: 21941-902. References 1. World Health Organization. Global status on Alcohol 2004. 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