© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

J Periodont Res 2016; 51: 95–102 All rights reserved

JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12287

Subgingival bacterial community profiles in HIVinfected Brazilian adults with chronic periodontitis

D. C. Ferreira1, L. S. Gonc ß alves1, 1 J. F. Siqueira Jr , F. L. Carmo2, H. F. Santos2, M. Feres3, L. C. Figueiredo3, G. M. Soares3, A. S. Rosado2, K. R. N. dos Santos2, A. P. V. Colombo2 1 Department of Endodontics and Molecular
cio de Sa
Microbiology Laboratory, Esta University, Rio de Janeiro, Brazil, 2Institute of
es, Federal Microbiology Prof. Paulo de Go University of Rio de Janeiro, Rio de Janeiro, Brazil and 3Dental Research Division, Department of Periodontology, Guarulhos University, Guarulhos, Brazil

Ferreira DC, Goncßalves LS, Siqueira JF Jr, Carmo FL, Santos HF, Feres M, Figueiredo LC, Soares GM, Rosado AS, dos Santos KRN, Colombo APV. Subgingival bacterial community profiles in HIV-infected Brazilian adults with chronic periodontitis. J Periodont Res 2016; 51: 95–102. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objective: To compare the subgingival microbial diversity between non-HIV-infected and HIV-infected individuals with chronic periodontitis using denaturing gradient gel electrophoresis (DGGE). Material and Methods: Thirty-two patients were selected: 11 were HIV-infected and 21 were non-HIV-infected, and all had chronic periodontitis. Periodontal measurements included probing depth, clinical attachment level, visible supragingival biofilm and bleeding on probing. Subgingival biofilm samples were collected from periodontal sites (50% with probing depth ≤ 4 mm and 50% with probing depth ≥ 5 mm) and whole-genomic-amplified DNA was obtained. The DNA samples were subjected to amplification of a 16S rRNA gene fragment using universal bacterial primers, followed by DGGE analysis of the amplified gene sequences. Results: The non-HIV-infected group presented higher mean full-mouth visible supragingival biofilm (p = 0.004), bleeding on probing (p = 0.006), probing depth (p < 0.001) and clinical attachment level (p = 0.001) in comparison with the HIV-infected group. DGGE analysis revealed 81 distinct bands from all 33 individuals. Banding profiles revealed a higher diversity of the bacterial communities in the subgingival biofilm of HIV-infected patients with chronic periodontitis. Moreover, cluster and principal component analyses demonstrated that the bacterial community profiles differed between these two conditions. High interindividual and intra-individual variability in banding profiles were observed for both groups. Conclusion: HIV-infected patients with chronic periodontitis present greater subgingival microbial diversity. In addition, the bacterial communities associated with HIV-infected and non-HIV-infected individuals are different in structure.

The periodontal microbiota is composed of a highly complex bacterial multispecies community organized in biofilms (1,2). Several studies have

investigated the composition of the periodontal microbiota in HIV-infected individuals (3–23). Interestingly, a higher prevalence of periodontal patho-

Lucio Souza Goncßalves, DDS, MSc, PhD,
cio de Sa
University, Faculty of Dentistry, Esta Av. Alfredo Baltazar da Silveira, 580/cobertura, Recreio, Rio de Janeiro, RJ, Brazil 22790-710 Tel: +55 21 24978988 Fax: +55 21 22566813 e-mail: [email protected] Key words: bacterial community profiling;

chronic periodontitis; denaturing gradient gel electrophoresis; HIV infections Accepted for publication April 5, 2015

gens has been demonstrated in non-HIV-infected than in HIV-infected individuals (10,17). However, microorganisms not commonly associated with

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periodontitis have frequently been detected in subgingival sites of HIVinfected patients, including Staphylococcus epidermidis, Candida albicans, Enterococcus faecalis, Clostridium difficile, Mycoplasma salivarium, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Entamoeba gingivalis (3,16,17,19,22,23). In particular, E. faecalis was more prevalent in the subgingival microbiota of HIV-infected patients with reduced levels of TCD4+ lymphocytes (< 200 cells/ mm3), suggesting that HIV-related immunodeficiency can provide appropriate conditions for the colonization and growth of opportunistic pathogens in the oral microbiota (16). Even though there are many studies on the periodontal microbiota of HIV-infected individuals, none have consistently profiled the overall bacterial community structures of the subgingival biofilm of these patients in comparison with the periodontal microbiota of non-HIV-infected individuals. The concept of community as the unit of pathogenicity has been proposed for several endogenous human infections and may certainly be applicable to periodontal diseases (1,2,24). This concept supports the notion that disease is a result of the synergism of microorganisms and the interaction of their products in a multispecies consortium (25). This piece of knowledge in HIV-infected individuals may provide additional insight into the ecology of periodontal diseases in this population, leading ultimately to the development of more effective therapeutic interventions. Several molecular methods have been used to profile bacterial communities of the periodontal microbiota, including pyrosequencing (26), terminal-restriction fragment length polymorphism (27) and denaturing gradient gel electrophoresis (DGGE) (28,29). The latter technique has been widely used in oral microbiology studies (30–33) and has the advantage of providing a picture of the community structure in the form of a pattern of bands. DGGE allows the simultaneous analysis of multiple samples, making it possible to compare the diversity of different communities.

The aim of the current study was to compare the subgingival microbial diversity between non-HIVinfected and HIV-infected individuals with chronic periodontitis using DGGE.

Material and methods Subject population

Thirty-two patients (11 HIV infected and 21 non-HIV infected) were selected for this study between 2010 and 2011. The HIV-infected group comprised patients who were attending the University Hospital Clementino Fraga Filho of the Federal University of Rio de Janeiro for HIV-infection monitoring and were referred for dental treatment. The non-HIV-infected patients were selected from a pool of first-time patients referred to the Periodontal Clinic of Guarulhos University for periodontal treatment. Of those, patients with chronic periodontitis (according to the clinical diagnosis described in the inclusion criteria) were selected during a period of a year. The inclusion criteria for all patients were as follows: diagnosis of chronic periodontitis; at least four sites with probing depth and/or clinical attachment level ≥ 5 mm; and bleeding on probing in different teeth. Patients were > 20 years of age and presented at least 15 teeth. Exclusion criteria included: need of antibiotic prophylaxis for dental procedures; pregnancy; diabetes; autoimmune diseases; necrotizing periodontal diseases; having used antibiotics and/or anti-inflammatory drugs in the last 6 mo; and periodontal therapy in the last 6 mo. All subjects were informed about the aims of the study and signed an informed consent to participate. The study protocol was approved by the Review Committee for Human Subjects of the University Hospital Clementino Fraga Filho. Clinical evaluation

Patients were asked to complete a dental and medical history questionnaire, and data on gender, age and means of HIV transmission were recorded. The

history of AIDS-defining opportunistic infections, TCD4+ lymphocyte counts, plasmatic HIV viral load and antiretroviral therapy were obtained from the patients’ medical records. Oral examination included visual inspection of the oral mucosa and periodontal evaluation. Periodontal measurements were recorded at six sites per tooth (distobuccal, buccal, mesiobuccal, distolingual, lingual and mesiolingual) in all teeth, excluding third molars, and included probing depth, clinical attachment level, bleeding on probing and visible supragingival biofilm. These measurements were performed by one calibrated examiner (intraclass correlation coefficient of 0.96 for probing depth and 0.97 for clinical attachment level) using a conventional manual periodontal probe (University of North Carolina, Hu-Friedy, Chicago, IL). After clinical examination, patients received full-mouth scaling and root planing under local anesthesia and instructions for proper home-care procedures. Microbiologic assessment collection— After removing the supragingival biofilm with sterile cotton pellets, subgingival biofilm samples were collected from four to nine periodontal sites (50% with probing depth ≤ 4 mm and 50% with probing depth ≥ 5 mm) of each individual using sterile Gracey curettes (Hu-Friedy), and the samples were immediately placed in separate microtubes containing 0.15 mL of 10 mM Tris–HCl, 1 mM EDTA, pH 7.6 (TE). Sample

DNA extraction— Biofilm samples were vortexed for 30 s and the microbial suspensions were washed three times with 100 lL of sterile Milli-Q water. Bacteria were pelleted by centrifugation at 2500 g, the pellet was resuspended in 100 lL of Milli-Q water and bacterial DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. DNA extracts were stored at
20°C until required for further analysis.

Subgingival microbial community in HIV infection Multiple displacement amplification—

DNA extracts from clinical samples were subjected to whole-genome amplification using the Illustra GenomiPhi V2 DNA Amplification kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer0 s instructions. In brief, 1 lL of DNA template was added to 9 lL of sample buffer containing random hexamer primers, denatured at 95°C for 3 min in a thermocycler and then cooled to 4°C. An aliquot of 1 lL of enzyme mix containing the phi29 DNA polymerase and additional random hexamers was mixed with 9 lL of reaction buffer containing deoxynucleotide triphosphates (dNTPs). This mixture was added to the denatured sample to a final volume of 20 lL and then incubated at 30°C for 1.5 h. The enzyme was then inactivated by incubation for 10 min at 65°C, and the amplified material was stored at
20°C. This multiple displacement amplification step was used to improve the performance of the subsequent PCR assays. PCR-DGGE

assay— A 16S rRNA

gene fragment of the whole-genomicamplified DNA extracts was amplified using the universal bacterial primers 968f [50 -AAC GCG AAG AAC CTT AC-30 ; containing a 40-bp GC clamp (50 -CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G-30 ) added to its 50 -end] and 1401r (50 -CGG TGT GTA CAA GAC CC-30 ). The presence of PCR products was confirmed by electrophoresis in a 1.5% agarose gel. The gel was stained for 15 min with 0.5 lg/mL of ethidium bromide and viewed under short-wavelength ultraviolet light. A 100-bp DNA ladder tool served as the molecular size standard. DGGE of the amplified gene sequences was performed using the Dcode Universal Mutation Detection System (Bio-Rad Dcode, Richmond, VA, USA) at 75 V and 60°C for 16 h in 1 9 Tris-acetate-EDTA. The PCR products (30 lL) were loaded onto 6% (wt/vol) polyacrylamide gels containing a linear gradient, ranging from 40% to 70%, of the denaturants urea and formamide [100% denaturant corresponded to 7 M urea and 40%

(v/v) formamide], and increasing in the direction of electrophoresis. The DGGE gels were stained with SYBR Gold (Invitrogen, S~ ao Paulo, SP, Brazil) and visualized using a Storm 860 Imaging System (GE Healthcare, Munich, Germany).

ware (GelCompar II Software, version 5.10; Applied Maths, Kortrijk, Belgium). Dendrograms for diverse comparisons of DGGE banding patterns were constructed with the unweighted pair group method using arithmetic averages (UPGMA). A similarity level of 60% was arbitrarily considered for cluster preview. The number of bands of each DGGE profile was calculated, and significant differences between groups were sought using the Mann– Whitney U-test. A matrix containing the presence or absence of each individual band in the samples was generated and used for the detrended correlation analysis (DCA), revealing the linear distribution of data (34). Clustering of samples was made by the principal component analysis, using, as input, the presence or absence of data. Shannon diversity indices were calculated for each DGGE profile analyzed using Canoco for Windows (Wageningen, the Netherlands) (35). Differences on demographic and periodontal clini-

Data analysis— Individual lanes of the

DGGE gel images were straightened and aligned using the GelCompar softTable 1. Immunological and HIV-related features of the HIV-infected group (n = 11 subjects) Value(mean  SD)

Variables +

a

TCD4 lymphocytes TCD8+ lymphocytesa TCD4+/TCD8+ HIV-infection exposure (years) HAART exposure (years)

618.0 979.4 0.7 10.7

   

97

296.5 459.5 0.3 6.8

7.6  3.2

a

Cells/mm3.HAART, highly active antiretroviral therapy.

Table 2. Demographic and periodontal clinical features of non HIV-infected and HIVinfected groups

Variables Age Gendera Male Female Periodontal parametersb Percentage of sites with: Visible supragingival biofilm Bleeding on probing Probing depth (mm) Clinical attachment level (mm)

HIV positive (n = 11)

HIV negative (n = 21)

47.3  6.9

45.0  9.1

0.271

7 (63.6) 4 (36.4)

10 (47.6) 11 (52.4)

0.410

58.0 34.8 2.7 3.0

   

15.1 21.4 0.4 0.6

75.8 63.4 3.7 4.0

   

13.3 27.9 0.5 0.7

p

0.004 0.006 < 0.001 0.001

Values are given as mean  SD or n (%).BOP, . a Mann–Whitney U-test. b Fisher’s exact test.

Fig. 1. Mean number of bands (A) and Shannon index (B) of bacterial communities of the subgingival biofilm of Control (non-HIV-infected) and HIV (HIV-infected subjects) with chronic periodontitis, determined by PCR-denaturing gradient gel electrophoresis (PCRDGGE) analysis. (p < 0.001, Mann–Whitney U-test).

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Ferreira et al.

Table 3. Frequency of denaturing gradient gel electrophoresis (DGGE) bands obtained from subgingival biofilm samples of 212 periodontal sites of the non-HIVinfected and HIV-infected groups HIV positive [n = 11 patients (66 sites)]

HIV negative [n = 21 patients (146 sites)]

Band

N

%

n

%

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17a B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30 B31 B32 B33 B34 B35 B36 B37 B38 B39 B40 B41 B42 B43a B44a B45a B46a B47a B48a B49 B50a B51 B52a

1 3 1 5 9 9 16 14 19 14 20 18 18 30 27 18 29 31 13 22 15 13 8 9 14 8 20 17 13 12 21 25 13 19 14 25 18 20 27 25 30 27 31 40 32 32 33 33 24 31 27 18

1.5 4.5 1.5 7.5 13.4 13.4 23.9 20.9 28.4 29.9 29.9 26.9 26.9 44.8 40.3 32.7 43.3 46.3 19.4 32.8 22.4 19.4 11.9 13.4 20.9 11.9 29.9 25.4 19.4 17.9 31.3 37.3 19.4 28.4 20.9 37.3 26.9 29.9 40.3 37.3 44.8 40.3 46.3 59.7 47.8 47.8 49.3 49.3 35.8 46.3 40.3 26.9

4 12 11 20 24 31 11 13 14 6 12 18 33 23 31 37 47 27 22 28 17 6 7 10 7 13 16 10 28 23 21 25 33 25 20 30 35 34 29 31 27 32 37 43 41 57 44 36 37 48 30 49

2.6 7.9 7.3 13.2 15.9 20.5 7.3 8.6 9.3 4.0 7.9 11.9 21.9 15.2 20.5 67.3 31.1 17.9 14.6 18.5 11.3 4.0 4.6 6.6 4.6 8.6 10.6 6.6 18.5 15.2 13.9 16.6 21.9 16.6 13.2 19.9 23.2 22.5 19.2 20.5 17.9 21.2 24.5 28.5 27.2 37.7 29.1 23.8 24.5 31.8 19.9 32.5

Table 3. (continued) HIV positive [n = 11 patients (66 sites)]

HIV negative [n = 21 patients (146 sites)]

Band

N

%

n

%

B53a B54 B55 B56 B57 B58 B59 B60 B61 B62 B63 B64 B65 B66 B67 B68 B69 B70 B71 B72 B73 B74 B75 B76 B77 B78 B79 B80 B81

30 21 27 12 27 25 24 16 7 20 10 3 4 7 1 2 4 3 2 3 3 6 2 3 5 9 5 3 4

44.8 31.3 40.3 17.9 40.3 37.3 35.8 23.9 10.4 29.9 14.9 4.5 6.0 10.4 1.5 3.0 6.0 4.5 3.0 4.5 4.5 9.0 3.0 4.5 7.5 13.4 7.5 4.5 6.0

41 35 36 21 23 27 32 21 11 15 14 13 4 4 3 4 1 4 2 2 3 4 3 7 0 2 0 5 1

27.2 23.2 23.8 13.9 15.2 17.9 21.2 13.9 7.3 9.9 9.3 8.6 2.6 2.6 2.0 2.6 0.7 2.6 1.3 1.3 2.0 2.6 2.0 4.6 0.0 1.3 0.0 3.3 0.7

a The most frequently detected bands in all samples (> 30%). Bold: difference of ≥ 20% in the frequency of bands between groups.

cal data between groups were examined with Fisher0 s exact and Mann– Whitney U-tests, using the SPSS software program (SPSS Statistics v. 19.0; IBM Brazil, S~ ao Paulo, SP, Brazil). Significance was established at 5% for all tests.

Results HIV-related characteristics

Table 1 shows the immunological and HIV-associated data of the HIVinfected group. All subjects had undetectable plasmatic HIV viral load (data not shown) and high mean levels of TCD4+ lymphocytes (618 cells/ mm3), suggesting that the disease was

under control in this group of subjects. In addition, all subjects were undergoing highly active antiretroviral therapy (HAART). Clinical and demographic features of the sample population

Information on demographic features and periodontal clinical parameters of both groups is presented in Table 2. No differences in gender and age were found between groups. In contrast, non-HIV-infected individuals presented significantly more visible supragingival biofilm, periodontal inflammation and tissue destruction compared with HIV-infected individuals. Microbiological data

Overall, DGGE analysis revealed 81 distinct bands from the 33 HIV- and non-HIV-infected individuals. Among the 212 subgingival sites analyzed, bands B17, B43–B48, B50, B52 and B53 demonstrated the highest frequency of detection (> 30%) (Table 3). The mean percentage of bands observed was significantly higher in the HIV-infected group (mean  SD: 16.6  7.1%) than in the non-HIV-infected group (mean  SD: 11.1  5.5%) (p < 0.001, Mann–Whitney U-test) (Fig. 1A). These results were also evident when the mean number of bands was calculated using the Shannon diversity index (Fig. 1B), indicating a higher diversity of bacterial communities in subgingival biofilm samples from the HIV group compared with the control group. High interindividual variability was also observed in terms of banding patterns (i.e. no two samples showed exactly the same bacterial community profile). Similar findings were observed regarding intra-individual analyses (data not shown). Comparative analysis of the two data sets revealed that the great majority of bands were present in both groups, and only two bands (B77 and B79) were exclusively found in HIV-infected patients (both were found in five sites) (Table 3). Cluster analysis revealed 33 different groups with similarity set at 60%

Subgingival microbial community in HIV infection

48.5%

HIV (+)

(16 clusters)

45.5% 6.0%

HIV (–)

(15 clusters)

Fig. 2. Distribution of 33 clusters of different denaturing gradient gel electrophoresis (DGGE) band profiles in non-HIV-infected [HIV (
)] and HIV-infected [HIV (+)] groups.

Site

Group HIV (+) HIV (+) HIV (+) HIV (+) HIV (+) HIV (+) HIV (+) HIV (+) HIV (+) HIV (+) HIV (+) HIV (+) HIV (+) HIV (+) HIV (+) HIV (–) HIV (–) HIV (–) HIV (–) HIV (–) HIV (–) HIV (–) HIV (–)

Fig. 3. Representative denaturing gradient gel electrophoresis (DGGE) profiles of PCRamplified bacterial 16S rRNA gene fragments from subgingival biofilm of eight non-HIVinfected [HIV (
)] and 15 HIV-infected [HIV (+)] subjects.

(Fig. 2). Only two clusters (6%) had samples from both groups; 48.5% and 45.5% of the clusters were exclusive to HIV-infected and non-HIV-infected groups, respectively. Figure 3 depicts the community profile of representative subgingival clinical samples obtained from eight non-HIV-infected and 15 HIV-infected subjects. Principal component analysis, based on the PCR-DGGE banding profile, showed a tendency of separation by PC1 axis of the samples from the nonHIV-infected subjects (Control) in relation to those from HIV-infected subjects (HIV) (Fig. 4). It can be observed that most of the samples from HIV-infected subjects were located in the right of the figure and

those from the non-HIV-infected samples in the left. In addition, samples from the non-HIV-infected subjects were closer to each other compared with samples from the HIV group. This indicates that the bacterial community is more similar among systemically healthy patients than among HIV-infected patients.

Discussion The current study compared the subgingival bacterial community structures of 11 HIV-infected patients with 21 non-HIV-infected subjects (all with chronic periodontitis) using DGGE analysis. The results showed higher diversity of the bacterial communities

99

in the subgingival biofilm of HIVinfected patients compared with nonHIV-infected individuals. There were also different community profiles between the groups, indicating the possibility of some patterns related to the HIV infection. Of interest, the periodontal clinical profile was less severe in HIV-infected patients than in non-HIV-infected subjects, corroborating data reported by other studies with the same population (17,18). One hypothesis that could partially explain these data is that HIV-infected individuals are normally more cautious about their general health and are exposed more to other oral/medical care professionals and other drugs, which might have an influence on the periodontal clinical parameters, such as the bleeding scores. Banding patterns of the subgingival microbiota in HIV- and non-HIV-infected individuals showed relative heterogeneity. The fingerprints in the HIV-infected group revealed more DGGE bands than those in the nonHIV-infected (control) group. The number of DGGE bands may be interpreted as the number of bacterial species in the community (36). Whereas some factors, such as as heteroduplex formation and discrepancies in the number of rrn operons in different species, may overestimate species richness, other factors may underestimate it, including sampling and sample homogenization biases, differential DNA extraction, PCR biases and co-migration on DGGE gels (37–39). The presence of a larger number of species composing the individual subgingival communities in the HIVinfected group suggests a more complex microbiota, despite the lower level of periodontal destruction observed in these patients. In addition, clustering analysis revealed that the subgingival samples clustered according to the clinical groups. This indicates that the bacterial communities associated with HIV-infected and non-HIV-infected individuals were different in structure (species composition and abundance). The high bacterial diversity of the oral microbiota of HIV-infected individuals has been previously reported

100

PC 2 5.1

0.8

Ferreira et al.

–0.6

SAMPLES Control –0.6

HIV 8.4

PC 1

1.0

Fig. 4. Score plot of principal component analysis based on PCR-denaturing gradient gel electrophoresis (DGGE) profiles of the total bacterial community in control (non-HIVinfected) and HIV (HIV-infected) individuals.

(17,40–42). Goncßalves et al. (17) observed that HIV-infected patients presented higher mean levels and prevalence of opportunist species involved in nosocomial infections, such as A. baumannii, E. faecalis and P. aeruginosa, in the subgingival biofilm than non-HIV-infected patients. Dang et al. (40) used microarray to compare the bacterial composition of the lingual microbiome between 12 HIV-infected patients (six untreated and six under HAART) and nine uninfected controls. The authors reported that the administration of antiretroviral therapy may change the profile of the oral microbiota, and indicated that chronic HIV infection may lead to substantial disruptions in the community structure of the lingual microbiota, even in the absence of clinical oral manifestations. Saxena et al. (41) utilized the DGGE method and also observed a greater diversity of the overall oral microbial population of HIV-positive individuals compared with HIV-negative controls. The higher bacterial diversity and the different community patterns in the periodontal microbiota of HIVinfected patients compared with nonHIV-infected individuals may be related to the HIV-infection condition, as well as to the long-term use of HAART and several other drugs

that adversely affect the oral immunity (43,44). For instance, it has been suggested that Notch-1 signaling can mediate epithelial differentiation in oral mucosa through interaction with TCD4+ lymphocytes (45,46). In this way, it is possible that depletion of TCD4+ lymphocytes from the oral mucosa of HIV-infected individuals may also lead to the impairment of epithelial growth and, accordingly, host–microbe dysbiosis (40). The reduced capacity of the oral immune response in this population has also been demonstrated by decreased expression of histatin-5 (a potent antimycotic agent) (47), tumor necrosis factor-a, interleukin-6 (44) and human b-defensin-2 (43). Several oral tissues, including gingiva (50), can express human b-defensin-2, which has a function of chemoattractant for dendritic cells and acts as part of the innate antimicrobial defense (48,49). Nittayananta et al. (43) demonstrated that the levels of human b-defensin-2 protein in saliva of HIV-infected individuals was increased with short-term use of HAART but decreased with long-term use of the medication. This change in oral mucosal immunity may result in significant alterations in the composition of oral bacteria, putting HIV-infected patients at risk of

developing oral infections (43). Iwai et al. (42) found distinct buccal and airway bacterial communities in HIVinfected patients with acute respiratory infections undergoing HAART and receiving antimicrobial therapy for pneumonia in comparison with non-HIV-infected controls. In summary, the results of this study showed a high interindividual variability in the bacterial composition of the subgingival microbiota. This is in agreement with the concept that chronic periodontitis may have a heterogeneous etiology and that periodontal destruction is the result of an ecological disruption of the microbiome, host response and periodontal microenvironment (1,51). However, several bands were shared by many of the DGGE profiles, suggesting that these species may be important keystone pathogens (52). It was not possible to identify these species after cutting out the DGGE bands and sequencing them because of the technical limitations, in some cases, and the poor quality of sequences obtained, even after cloning (data not shown). In conclusion, the microbiota associated with periodontitis in HIVpositive individuals was shown to differ significantly in comparison with that of HIV-negative individuals. Obtaining a better understanding of the microbial composition of the periodontal microbiota, and its complex interaction with the host, has been a great challenge for numerous investigators over the years. Understanding these mechanisms is even more challenging when many other factors are present, as seen in HIV-infected patients. In these individuals, an unbalanced periodontal microbial community as a result of local or systemic conditions can be observed. The complexity of this microbial community needs to be further unraveled in order to provide information regarding the possible interaction with other oral sites and the systemic repercussion.

Acknowledgements This study was supported by grants from National Council for Scientific

Subgingival microbial community in HIV infection and Technological Development (CNPq), Brasilia, Brazil; 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.

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