Oral Bacteriome of HIV‐1‐Infected Children from Rio De

PROF. LUCIO SOUZA GONÇALVES (Orcid ID : 0000-0002-4388-6310)

Article type : Original Article Clinical Periodontology

Oral bacteriome of HIV-1-infected children from Rio de Janeiro – Brazil: Next- generation DNA sequencing analysis

Running title: Oral bacteriome of HIV-infected children

1  Lucio Souza Gonçalves, DDS, MSc, PhD 1,2  Dennis de Carvalho Ferreira, DDS, MSc, PhD 7  Nicholas C. K. Heng, BSc, PhD 1  Fabio Vidal, DDS, MSc, PhD 3  Henrique Fragoso dos Santos, DDS, MSc, PhD 7  Diogo Godoy Zanicott, BDS, MClinDent, PhD 4  Mariana Vasconcellos, DDS, MSc 2  Mayra Stambovsky, DDS, MSc 8  Blair Lawley, PhD 6  Norma de Paula Motta Rubini, PhD 5  Katia Regina Netto dos Santos, MSc, PhD 9  Gregory J Seymour, BDS, MDSc, PhD

1Programa de Pós-Graduação em Odontologia, Universidade Estácio de Sá, Rio de

Janeiro, Brazil. Author Manuscript

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/JCPE.13176

3Universidade Federal Fluminense, RJ, Brazil.

4 Universidade Federal do Rio de Janeiro, Rio de janeiro, Brazil.

5Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

6Departmento de Imunologia, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, Brazil.

7Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand

8Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

9School of Dentistry, The University of Queensland, Brisbane, Australia

Conflict of interest: The authors declare that they have no competing interests.

Acknowledgements

This work was supported by National Council for Scientific and Technological Development (CNPq) (Project: 211309/2013-3) and Foundation for Research Financial Support in the State of Rio de Janeiro (FAPERJ), Rio de Janeiro, Brazil (Project: E26/103.001/2012).

Correspondence and reprints should be sent to:

Lucio Souza Gonçalves, Estácio de Sá University, Rio de Janeiro, Brazil

Address: Av. Alfredo Baltazar da Silveira, 580 – cobertura, Recreio dos

Bandeirantes, Rio de Janeiro, RJ, Brazil.

Postal code: 22790-710Author Manuscript

Telephone number: +5521994550040

Email: [email protected]

Background & Aim: This study compared the oral bacteriome between HIV-1-infected and non-HIV-1-infected Brazilian children/teenagers.

Methods: Whole saliva, biofilm from the dorsal surface of the tongue, and biofilm from supragingival and subgingival sites were collected from 27 HIV-1-infected and 30 non- HIV-1-infected individuals. Bacterial genomic DNA was extracted and 16S rRNA genes were sequenced using next-generation sequencing technology (Ion Torrent).

Results: In the supragingival biofilm, the phylum Firmicutes and genus Streptococcus sp. were more frequent in HIV-1-infected (95% and 78%, respectively) than in non- HIV-1-infected individuals (40% and 24%, respectively). In the subgingival biofilm of HIV-infected participants, the relative abundance of the Veillonella sp. and Prevotella sp. genera were higher than in non-HIV-1-infected participants. On the tongue, the genera with greater relative abundance in HIV-1-infected individuals were Neisseria sp. (21%). In saliva, the difference of the genus Prevotella sp. between non-HIV-1-infected and HIV-1-infected individuals was 15% and 7%, respectively. The Chao index revealed an increase in the richness of both sub- and supragingival biofilms in the HIV- 1-infected samples compared with non-HIV-1-infected samples.

Conclusion: HIV-1-infected children/teenagers have a higher frequency of the phyla Firmicutes and genus Streptococcus, and their oral microbiome shows more complexity than that of non-HIV-1-infected children/teenagers.

CLINICAL RELEVANCE

Scientific rationaleAuthor Manuscript for study

Only two papers have been published regarding the oral microbiome of HIV-1-infected children.

The samples of supragingival dental biofilm of HIV-1-infected subjects were almost entirely dominated by Firmicutes (~95%). Neisseria sp., Leptotrichia sp. and Fusobacterium sp. with relative abundance of approximately 13%. 15% and 8% in non- HIV-1-infected individuals respectively, were not found in HIV-1-infected children/teenagers.

Practical implications

Oral bacterial community shifts or dysbiosis are relevant factors associated with oral diseases, such as caries and periodontal disease, as well as with systemic events in HIV- 1-infected children/teenagers.

INTRODUCTION

The human immunodeﬁciency virus-1 (HIV-1) epidemic has been so dramatic and devastating that it has been described as the “epidemic of our century” (Merchant et al. 2001). It is estimated that approximately 36.7 million people, (2.1 million children younger than 15 years old) are currently living with HIV-1. (UNAIDS/WHO – Global HIV epidemic, 2017).

Several studies have assessed the periodontal microbiota of HIV-1-infected patients (Zambon et al. 1990, Murray et al. 1991, Luch et al. 1991, Rams et al. 1991, Moore et al. 1993, Cross & Smith 1995, Brady et al. 1996, Tenenbaum et al. 1997, Scully et al. 1999, Jabra Risk et al. 2001, Tsang & Samaranayake 2001, Patel et al. 2003, Gonçalves et al. 2004, Gonçalves et al. 2007, Aas et al. 2007, Gonçalves et al. 2009, Ramos et al. 2012). Nevertheless, the findings of these studies are still controversial. Studies have detected a greater prevalence of periodontal pathogens such as Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, and Treponema denticola, as

well as a combinationAuthor Manuscript of these species in HIV-1-infected patients compared with non- HIV-1-infected individuals (Cross & Smith 1995, Tenenbaum et al. 1997, Scully et al. 1999, Patel et al. 2003). Microorganisms usually not considered as members of the periodontal microbiota have also been identified in HIV-1-infected individuals, such as

This article is protected by copyright. All rights reserved Staphylococcus epidermidis, Candida albicans (Zambon et al. 1990, Odden et al. 1994, Chattin et al. 1999), Enterococcus faecalis, Clostridium clostridiiforme, Clostridium difficile, Mycoplasma salivarium (Zambon et al. 1990, Moore et al. 1993, Gonçalves et al. 2004, Gonçalves et al. 2007), Acinetobacter baumannii and Pseudomonas aeruginosa (Gonçalves et al. 2007).

Very few studies regarding the oral microbiota of HIV-1-infected children have been reported and most of them are usually restricted to Candida species (Brown et al. 2000, Munro & Hube 2002, Bosco et al. 2003, Pongsiriwet et al. 2004, Portela et al. 2004). Differences in the distribution of cariogenic microorganisms, such as Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus acidophilus, have not been observed between HIV-1 and non-HIV-1-infected children (Castro et al. 2004). However, Silva-Boghossian et al. (2007) have shown higher frequencies and levels of beneﬁcial species of oral streptococci, together with Actinomyces sp., and Veilonella parvula, potential oral pathogens (Eubacterium nodatum, F. nucleatum, Parvimona micra, P. intermedia, Selenomonas noxia, T. forsythia, T. denticola) and species not usually detected in the oral cavity (Bacillus cereus, Corynebacterium diphteriae, E. faecalis, Klebsiella pneumoniae, P. aeruginosa, and Staphylococcus aureus) in the salivary microbiota of non-HIV-1 than HIV-1-infected children.

A number of authors have studied the oral microbiota of HIV-1-infected individuals using gene amplicon sequencing (Dang et al. 2012, Li et al. 2014, Beck et al. 2015, Kistler et al. 2015, Noguera-Julian et al. 2017, Presti et al. 2018, Goldberg et al. 2015, Starr et al. 2018), however only two papers have been published regarding the oral microbiome of HIV-1-infected children (Goldberg et al. 2015, Starr et al. 2018). These studies were carried out with HIV-1-infected children from the USA, by the use of 454 pyrosequencing (Goldberg et al. 2015) or Illumina platform on a MiSeq (Starr et al. 2018).

The relationship between the oral microbiome and oral disease in HIV-1- infected patients is still poorly understood, especially in children (Goldberg et al. 2015,

Starr et al. 2018).Author Manuscript It has been proposed that HIV-1-infection is associated with alterations in the gut microbiome. These occur early in the course of infection, as a consequence of the depletion of CD4+ T cells in the intestinal mucosa and are not fully restored with antiretroviral therapy (Brenchley et al. 2006, Zevin et al. 2016, Saxena

This article is protected by copyright. All rights reserved et al. 2016, Heron & Elahi 2017). Whether similar changes occur in the oral microbiome are, as yet, unknown. The aim of the current study therefore was to compare the oral bacteriome between non-HIV-1 and HIV-1-infected Brazilian children/teenagers using Ion Torrent 16S rRNA gene amplicon sequencing.

MATERIAL AND METHODS

Subject selection

Twenty-seven patients were selected from a group of the HIV-1-infected children/teenagers between 2 and 18 years old who were in outpatient follow up at the Department of Pediatric Immunology, University Hospital Gaffrée and Guinle at the Federal University of Rio de Janeiro State (HUGG/UNIRIO), Brazil. Thirty HIV-1- sero-negative children/teenagers were selected from the Pediatric Dentistry Clinic of the Faculty of Dentistry at Estácio de Sá University. The parents or guardians of all children/teenagers were informed in writing about the objective of the study, its risk and benefits, and a signed consent obtained. Following consent, a medical history including demographic data as well as medical and dental information was obtained from the parents or guardians. Exclusion criteria included presence of diabetes, cardiovascular diseases, cancer, chronic inflammatory and/or autoimmune diseases, use of antibiotics and/or anti-inflammatory drugs in the past 6 months prior to initial evaluation.

Data regarding clinical manifestations as well as the use of medications and laboratory parameters, including levels of TCD4 lymphocyte, TCD8 lymphocytes and viral load were obtained from the patient’s medical record. Oral examination included visual inspection of the oral mucosa and periodontal evaluation. Periodontal measurements were recorded at six sites per tooth (disto-buccal, buccal, mesio-buccal, disto-lingual, lingual and mesio-lingual) and included bleeding on probing (BOP) and visible supragingival biofilm (VSB).

Ethical aspects

The studyAuthor Manuscript was developed in accordance with the 1975 Helsinki Declaration, updated in 2013. The experimental protocol was approved by the Ethics Committee of the Estácio de Sá University, RJ, Brazil (CAAE: 14356813.0.0000.5284).

Definition of the groups

Children/teenagers considered HIV-1-infected were seropositive for the HIV-1 by ELISA and confirmed by Western blot. Definition of the non-HIV-1-infected Children/teenagers considered non-HIV-1-infected were declared as such by their parents or guardians. Microbiological assessment In order to assess the diversity of the oral bacteriome, samples of whole saliva (HIV-1-infected: n = 15 individuals; non-HIV-1-infected: n = 26 individuals) and biofilm from the dorsal tongue (HIV-1-infected: n = 8 individuals; non-HIV-1-infected: n = 12 individuals) were collected. In addition, pooled samples of supragingival (HIV- 1-infected: n = 6 individuals; non-HIV-1-infected: n = 11 individuals) and subgingival biofilm (HIV-1-infected: n = 7 individuals; non-HIV-1-infected: n = 11 individuals) from 12 teeth (two teeth of each sextant) were also collected. The saliva samples were collected by spitting into a sterile collector for three times. The dorsal tongue biofilm was collected using a sterile swab rubbed twice and then inserted into a sterile collector containing TE solution (Tris-EDTA, pH 7.4). Supragingival biofilm samples were collected from the mesiobuccal sites of teeth number 51/11, 53/13, 55/15, 61/21, 63/23, 65/25, 71/31, 73/33, 75/35, 81/41, 83/43, and 85/45 (FDI numbering system) utilizing sterile periodontal curettes and pooled. Samples of the subgingival biofilm were also collected from the same sites and pooled. DNA extraction, 16S rRNA gene sequencing and bioinformatics analysis Genomic DNA was extracted using the PowerSoil DNA Isolation kit (MoBio Laboratories. Carlsbad. CA. USA) according to the manufacturer’s instructions. DNA concentration was determined by a Qubit fluorometer (Invitrogen. Carlsbad. CA. USA). Bacterial 16S rRNA gene amplicons of variable regions V1-V2 were amplified using the primers 27F (AGAGTTTGATCMTGGCTCAG) and 357R (CTGCTGCCTYCCGTA), tagged with the Ion Torrent adapter sequences and MID barcode. Polymerase chain reactions (PCR) were performed according to the manufacturer’s Author Manuscript protocols. Sequencing was performed on an Ion Torrent Personal Genome Machine following the manufacturer’s protocols (LGC Genomics. Berlin. Germany).

This article is protected by copyright. All rights reserved The raw sequence data was processed in QIIME v.1.9.1 (Caporaso et al. 2010). Adapter and barcodes were removed from each sequence. Reads shorter than 200 bp were removed. Sequences were filtered using an average cutoff of Q20. Chimeras were removed using UCHIME. The operational taxonomic units (OTUs) were constructed with a 3% dissimilarity. For all OTU-based analyses, the original OTU table was rarified to a depth of 1,800 sequences per sample to minimize the effects of sampling effort on the analysis. Taxonomic identities were assigned for representative sequences of each OTU using the Greengenes (version 13_8) database (DeSantis et al. 2006). The clusters were used for generating predictive rarefaction models and for determining non-parametric species-richness estimators, such as abundance-based coverage estimators Chao1 (Chao and Bunge 2002), and the Shannon diversity index. Statistical analysis Statistical software (Statistical Package for the Social Sciences 21.0; IBM, Armonk, NY) was used for all statistical analyses. The normality of the quantitative variables was checked using the Kolmogorov-Smirnov test and graphic analysis. The continuous variables were described as mean [standard deviation (SD)] and median (range), and categorical characteristics as frequency. The difference between groups was compared using the Mann-Whitney U test for continuous variables and the chi square test or Fisher exact test for categoric variables. The relative abundance at the phylum and genera taxonomic levels were calculated for each patient, then average (SD) and median (range) obtained within each clinical group for each kind of sample (saliva, biofilm from the dorsal tongue, supragingival and supragingival biofilm). In the comparisons done at each taxonomical level, the Benjamini–Hochberg (1990) method was used for adjustment of multiple comparisons error (an overall adjusted p-value < 0.0027 was considered significant), beyond the effect size calculation [effect size (d): large = 0.80, medium = 0,50 and small = 0,20] for the U de Mann-Whitney test (Cohen 1992). The level of significance established in all analyses was 5% (p < 0.05).

RESULTS The characteristics of the 27 HIV-1-infected children/teenagers are presented in

Tables 1 and 2Author Manuscript . The age ranged from 3 to 18 years, and the frequency of females was 55.6 % (15/27). The range of TCD4+ and TCD8+ lymphocytes was 60 – 1803 cells/mm3 and 747 - 3368 cells/mm3, respectively. All patients were under HAART; however five patients did not follow the treatment schedule.

This article is protected by copyright. All rights reserved Comparison between non-HIV-1-infected and HIV-1-infected children/teenagers is shown in Table 2. Only the variable “dental floss use” showed a significant difference between groups (p = 0.001). In both groups, no individual presented with periodontitis.

Bacterial community The 16S rRNA gene sequencing of oral samples of non-HIV-1-infected and HIV-1-infected children/teenagers revealed a total of 12 phyla, 18 classes, 25 order and 63 genera. Approximately 96% of the sequences of all samples belonged to 4 phyla. The most represented phyla were Firmicutes (~58%), Fusobacteria (~14%), Proteobacteria (~13%) and Bacteroidetes (~11%) (Figure 1). The most represented classes were Bacilli (~41%), Fusobacteria (~15%), Negativicutes (~12%), Bacteroidia (~11%) and Betaproteobacteria (~9%) (Appendice 1). The most dominant orders were Lactobacillales (~42%), Fusobacteriales (~14%), Selenomonadales (~13%), Bacteroidales (~9%) and Neisseriales (~9%) (Appendice 2). And approximately 74% of the sequences belonged to only 5 genera: Streptococcus (~37%), Veillonella (~12%), Neisseria (~9%), Leptotrichia (~9%) and Prevotella (~7.5%) (Figure 2)

The phylum Firmicutes showed a higher relative abundance in all samples of HIV-1-infected individuals, with the exception of those from dorsal tongue. The samples that showed the greatest difference were the ones from supragingival dental biofilm (Figure 1). In the samples of supragingival dental, there were no statistically significant differences between groups in terms of the mean relative abundance for these phyla (p > 0.0027, adjustment of multiple comparisons error), however the effect size (d) presented large value (d ≥ 0.8), characterizing a strong magnitude of the effect in those comparisons (Table 4). In the HIV-1-infected group, the mean relative abundance of Firmicutes, Fusobacteria and Proteobacteria were 95.4% (standard deviation = 3.1), 0.38% (SD = 0.40) and 0.65% (SD = 0.49) respectively while in the non-HIV-1-infected group the mean relative abundance of these phyla were 41.5% (SD = 7.6), 25.2% (SD = 24.0) and 18.9% (SD = 21.9), respectively (p = 0.003, d = 2.144; p = 0.004, d = 1.999; p

= 0.027, d = 1.271,Author Manuscript respectively). Indicating an increase of Firmicutes and a decrease of Fusobacteria and Proteobacteria in the samples of supragingival dental biofilm in HIV- 1-infected individuals.

This article is protected by copyright. All rights reserved At the genus level, the highest variation was also observed in the samples of supragingival dental biofilm (Figure 2). The genus Streptococcus sp. showed a higher relative abundance in the samples of HIV-1-infected individuals (78%). The differences in the mean relative abundance for these genera were statistically significant between the groups only for Streptococcus sp.: HIV-1-infected 77.8% (SD = 9.30) and non-HIV- 1-infected 24.2% (SD = 15.8) (p = 0.001; d = 2.708). Although Neisseria sp., Leptotrichia sp. and Fusobacterium sp. did not demonstrate significant difference between groups [HIV-1-infected 0.28% (SD = 0.25), 0.26% (SD = 0.33) and 0.09% (SD = 0.09), respectively; non-HIV-1-infected, 12.2% (SD = 14,9), 16.3% (SD = 15.4) and 8.61% (SD = 9.70), respectively; p > 0.0027, adjustment of multiple comparisons error], the magnitude of the effects was large (d = 1.271, d = 2.144, d = 1.748, respectively) (Table 4).

The samples of subgingival dental biofilm showed higher relative abundance of genera Veillonella sp. and Prevotella sp. in HIV-1-infected individuals (23% and 10%, respectively) when compared with non-HIV-1-infected individuals (11% and 5%, respectively) (Figure 2), however the comparison of the mean relative abundances between the groups was not statistically significant (p > 0,05) (Table 4). Conversely, on the dorsal tongue, these genera presented a greater relative abundance in non-HIV-1- infected individuals (approximately 24% and 16%, respectively) than in HIV-1-infected individuals (10% and 3%, respectively) (Figure 2). These differences in the mean relative abundances demonstrated a large effect size [Veillonella sp. (d = 1.266) and Prevotella sp. (d = 1.210): HIV-1-infected 10.0% (SD = 18.2) and 3.30% (4.64), respectively; non-HIV-1-infected 23.8% (SD = 19.8) and 15.6 (13.1), respectively), although there were no statistically significant (p > 0.0027, adjustment of multiple comparisons error) (Table 4). The genera with greater relative abundance in the dorsal tongue of HIV-1-infected individuals were Neisseria sp. (21%) and Leptotrichia sp. (15%), but the mean relative abundances of these genera did not show significant difference when compared with non-HIV-1-infected individuals (p > 0,05) (Table 4). In the saliva, the genus Prevotella sp. showed a medium effect size (d = 0.739) when non-

HIV-1 and HIV-1Author Manuscript individuals were compared (approximately 15% and 7%, respectively), although the p value adjusted > 0.0027 (Figure 2 and Table 4).

The Chao index revealed an increase in the richness of sub and supragingival dental biofilm in HIV-1-infected samples, when compared with non-HIV-1-infected

This article is protected by copyright. All rights reserved samples (Table 3). The Shannon diversity index, which takes into account the species richness and evenness, revealed that there are no differences between HIV-1 and non- HIV-1-infected individuals, with the exception of supragingival biofilm samples. In these samples, the value was higher in HIV-1-infected children (Table 3).

The principal component analysis (PCA) show different patterns depending on the site evaluated (Figure 3). The dental biofilm samples, mainly supragingival biofilm, show high similarity between the non-HIV-1-infected individuals. The supragingival samples appeared not to have any impact on the bacterial community with all samples showing a high degree of similarity, with the exception of three individuals. The subgingival biofilm samples, however, did show a small difference between HIV-1 and non-HIV-1-infected individuals. Within each group the HIV-1-infected samples showed more variation compared with most of non-HIV-1-infected which were highly similar. Although dorsal tongue samples did not demonstrate a high degree of similarity, there was a higher concordance between HIV-1-infected samples than between non-HIV-1- infected samples. The differences in the bacterial community on the dorsal tongue between individuals appears to be due to the HIV-1-infection. The saliva samples showed the higher difference between the individuals, but this did not reveal any impact due to HIV-1-infection.

DISCUSSION

The current study analyzed the oral bacterial community of HIV-1-infected and non-HIV-1-infected Brazilian children/teenager, using next-generation sequencing of 16S rRNA gene amplicons (Ion Torrent). As there is a lack of studies assessing the oral microbiome in HIV-1-infected children, (Goldberg et al. 2015, Starr et al. 2018), it is relevant to carry out this type of research in order to understand the characteristics of the oral microbiome in this population. The findings demonstrated higher bacterial richness (Chao-1) of supragingival and subgingival biofilm in HIV-1-infected individuals compared with non-HIV-1- infected children/teenagers, while the Shannon diversity index was higher in HIV-1-

infected only forAuthor Manuscript supragingival biofilm. (Table 3). Studies with HIV-1-infected adults using different microbiological methods, such as microarray (Dang et al. 2012) and DGGE (Saxena et al. 2012, Ferreira et al. 2016), also demonstrated higher microbial diversity in HIV-1-infected when compared with non-HIV-1-infected individuals.

This article is protected by copyright. All rights reserved The results of the present study are in conflict with those of Goldberg et al. 2015, Noguera-Julian et al. 2017 and Starr et al. 2018. The reason for this apparent conflict could lie in the fact that different populations were studied.

For all samples, in both groups, the most frequent phyla were Firmicutes, followed by Fusobacteria, Proteobacteria and Bacteroidetes. Goldberg et al. (2015) and Noguera-Julian et al. (2017) found similar results, except that the phyla Proteobacteria did not appear with high prevalence in the Goldberg et al. (2015) study. Of interest, the phyla Firmicutes showed a higher relative abundance in HIV-1-infected children, with approximately 95% of the total bacterial community. The study of Li et al. (2014), using microarray and pyrosequencing molecular techniques to analyze the salivary microbial composition of 10 HIV-1-infected, before and after HAART, and 10 non- HIV-1-infected adults from the USA, demonstrated a different bacterial profile among the three groups enrolled in the investigation, with the highest frequency of phylum Firmicutes (65%). In fact, it has been hypothesized that HIV-1 infection disrupts the intestinal immune system, leading to microbial translocation and systemic immune activation, and that the same effects may impact the oral mucosa/saliva immune activity (Saxena et al. 2016). However, HAART may shift the oral microbiome to more closely resemble the non-HIV-1-infected group (Saxena et al. 2016), thus, the composition found in the current study may be due to the fact that all HIV-1-infected children were under HAART. In addition, Starr et al. (2018) speculated that in adolescents whose HIV-1-infection is controlled by HAART or other therapies, the oral microbiota would be similar to that of non-HIV-1-infected, and the microorganisms that cause oral disease would also be similar. Even though, Presti et al. (2018), in a cohort study with HIV-1- infected adults, did not find changes in the salivary microbiome after 24 weeks of HAART, with Bacteroidetes, Firmicutes and Proteobacteria remaining the more abundant phyla.

At the genus level, Streptococcus sp. showed a higher relative abundance in the samples of supragingival dental biofilm of HIV-1-infected individuals (77.8%) compared with non-HIV-1-infected individuals (24.2%). Kistler et al. (2015) also Author Manuscript demonstrated a predominance of the genus Streptococcus in supragingival biofilm of HIV-1-infected. Despite the fact that supragingival sites of HIV-1-infected children showed an increase in the relative abundance of Firmicutes phylum and Streptococcus genus, the diversity and richness indexes were higher. This can be explained by the high

This article is protected by copyright. All rights reserved number of different OTUs belonging to the genus Streptococcus sp. This genus showed high relative abundance in all sites studied. The most abundant OTUs of this genus showed similarity with sequences belonging to uncultured Streptococcus, S. parasanguinis, S.mitis and S. salivarus. Species of this genus are usually detected in the oral cavity and are considered the main group of early colonizers (Kreth at al., 2009).

In the current study, the relative abundance of Neisseria sp., L eptotrichia sp. and Fusobacterium sp. was approximately 12%, 16% and 9% in supragingival sites of non-HIV-1-infected children, respectively; these were not found in HIV-1-infected. These genera are important members of a healthy human oral cavity (Takeshita et al. 2016), and the loss of them reveals a dysbiosis of the bacterial community, possibly associated with HIV-1.

Neisseria is a member of the healthy ‘core microbiome’ of the human oral cavity (Takeshita et al. 2016). Neisseria human commensalism appears to be conserved across different geographical regions, ethnic groups and lifestyles (Liu et al, 2015). However, the current study showed the loss of this important genera in HIV-1-infected children. However, in contrast with the current study, Noguera-Julian et al. (2017) showed Neisseria sp. to be 4-fold higher in HIV-1-infected individuals than in non- HIV-1-infected individuals. This finding may have been due to the fact that adults with periodontitis were studied.

This reduction of some genera in the supragingival biofilm may be associated with the HAART. For example, some oral opportunistic infections are rarely seen in well-controlled HIV-1-positive patients, with the oral microbiota of well-controlled HIV-1-infected patients presenting essentially the same as non-HIV-1-infected patients. In fact, in the current study, 22 of the 28 HIV-1-Infected children had CD4+ T lymphocytes counts above than 500 cells/ mm3. This suggests that a shift in the oral microbiome in these patients may have occurred after HAART (Saxena et al. 2016).

The samples of subgingival dental biofilm showed higher relative abundance of genera Veillonella sp. and Prevotella sp. in HIV-1-infected individuals (23% and 10%, Author Manuscript respectively) when compared with non-HIV-1-infected individuals (11% and 5%, respectively). Oral Veillonella species, especially V. parvula, have been associated with severe early childhood caries (Kanasi et al. 2010) and are also often found in the subgingival biofilm from patients with chronic periodontal diseases (Heller et al. 2012,

Starr et al. 2018 observed that in a sample of children from the USA, with perinatally HIV-1-infection, the genus Streptococcus sp. represented over 65% of the total taxa detected in the subgingival biofilm. Other abundant taxa included Granulicatella sp., Fusobacterium sp. and Haemophilus sp. Further, Goldberg et al. (2015) showed that in patients with poor oral hygiene the subgingival biofilm contained the genera Gemella sp., Neisseria sp. and Porphyromonas sp. Although both studies (Starr et al. 2018, Goldberg et al. 2015) used HIV-1-infected children, the findings were completely different when compared with the current study. This divergence of results may reflect the different populations studied. In addition, both HIV-1-infected and non-HIV-1-infected individuals in the current study had good plaque control with a low VSB.

In the saliva, the genus that showed a difference when comparing non-HIV-1 and HIV-1-infected individuals, was Prevotella sp., with approximately 15% and 7% abundance respectively. One study with HIV-1-infected children (Goldberg et al. 2015) and three with HIV-1-infected adults (Kistler et al. 2015, Presti et al. 2018, Noguera-Julian et al. 2017) analyzed the salivary microbiome but none demonstrated findings similar with the current study. These different findings may again reflect population differences among the studies.

In the tongue, the genera with greater relative abundance in non-HIV-1-infected individuals, compared with HIV-1-infected, were Veillonella sp. and Prevotella sp. and in HIV-1-infected individuals, the genera with greater relative abundance were Neisseria sp. and Leptotrichia sp. The comparison of these findings with other studies is not possible because no previous study has analyzed the dorsal tongue biofilm of HIV- 1-infected children.

The PCA analysis revealed different patterns according to the evaluated site. The dental biofilm samples showed higher similarity among different individuals compared Author Manuscript with dorsal tongue biofilm and saliva samples. The bacterial community of tongue and principally saliva showed a higher fluctuation among individuals. The impact of HIV-1- infection on the bacterial community patterns was unclear, and the sites that appear to show a small change were subgingival and tongue. This is in agreement with Dang et

This article is protected by copyright. All rights reserved al. 2012, who, also showed an impact of the HIV-1-infection on the dorsal tongue microbiota, albeit in untreated HIV-1-infected adults. The effect of HIV-1-infection in the subgingival and tongue sites could be associated with the CD4+ T cell depletion and consequence decrease of T cell associated cytokines, including IL-17 and IL-23, which play a role in the protection against mucosal candidiasis (Huppler et al. 2014) and other opportunistic infections (Heron & Elahi 2017). In addition, Notch-1 signaling mediates oral epithelial cell differentiation (Casey et al. 2006) and regulates the activity of CD4+ T cell responses by promoting cellular longevity (Helbig et al. 2012). The hypothesis is that CD4+ T cell reduction from the subgingival and tongue epithelium of HIV-1-infected individuals can affect the epithelial growth, disrupting the oral barrier, with consequence microbial dysbiosis (Dang et al. 2012), microbial translocation and systemic immune activation (Saxena et al. 2016). The difference in bacterial communities between HIV-1-infected individuals appears to result in an increase subgingivally and a decrease on tongue.

In summary, the findings of the current study suggest a bacterial dysbiosis in the HIV-1-infected Brazilian children/teenagers. This dysbiosis was seen predominantly within the dental biofilm (both supra and subgingival), with the apparent loss of important organisms, such as Neisseria sp., Leptotrichia sp. and Fusobacterium sp., and a concomitant increase in Veillonella sp. and Prevotella sp. In addition, HIV-1 infected children/teenagers appear to have a high frequency of the phylum Firmicutes and the genus Streptococcus sp., in their supragingival biofilm (~95% and 78%, respectively). In fact, HIV-1 infected Brazilian children/teenagers appear to have an oral microbiome with higher complexity than non-HIV-1 infected individuals. This may be due to several factors, such as the regular use of antimicrobials to treat recurrent infections, HAART and modification of immune response. Future studies using the same methodology are required in order to confirm these characteristics in this population. In addition, this study has a potential limitation that was not able to evaluate the caries experience of the children. In this way, clinical longitudinal studies are needed to understand bacterial community shifts or dysbiosis and its relationship with oral diseases, such as caries and

periodontal disease,Author Manuscript as well as with systemic events

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This article is protected by copyright. All rights reserved Table 1. Characteristics of HIV-1-Infected children/teenagers Patient ID Age Gender Clinical classification Antiretroviral therapy CD4 CD8 HIV Viral load

1 18 Male NI EFZ/ddI 843 1823 UND

2 5 Female A1 AZT/3TC/NVP 1250 1845 UND

9 12 Female A3 AZT/3TC/EFZ 601 747 UND

10 14 Male B3 AZT/3TC/NVP 588 1048 699

35 13 Female NI NI 1250 1845 UND

37 5 Female A1 AZT/3TC/NVP 1803 1608 UND

38 12 Female A3 AZT/3TC/EFZ 60 748 UND

39 6 Female C3 AZT/3TC / Kaletra NI NI NI

40 9 Male NI AZT/3TC/EFZ 668 1920 833

41 8 Female NI NI 588 1048 699

42 9 Male A2 NI 1031 774 UND Author Manuscript

44 13 Female B3 3TC/NVP/TDF NI NI NI

45 8 Male A2 AZT/3TC/Kaletra 1031 874 100

46 9 Female NI AZT/ 3TC/ Kaletra 1055 783 UND

47 14 Male NI 3TC/TDF/ Kaletra 815 3368 UND

48 14 Male C3 AZT/3TC/TDF/EFZ/Kaletra 263 2194 16184

49 12 Male NI AZT/3TC/EFZ 1108 1407 250

50 13 Male C3 3TC/TDF/Kaletra 104 1371 UND

51 13 Male C3 3TC/TDF/ Kaletra 978 832 UND

52 6 Female C3 AZT/3TC/ Kaletra NI NI NI

53 13 Female C3 3TC/EFZ/TDF/ 1365 858 UND

54 NI Female NI NI 799 1011 UND

55 3 Female B2 AZT/3TC/ Kaletra 669 1503 NI Author Manuscript

57 13 Female B1 AZT/3TC/NVP 1250 1845 UND

58 8 Female C3 AZT/Kaletra/ddI 601 747 UND

NI: no information; UND: undetectable; AZT: Zidovudine; EFZ: Efavirenz; NVP: Neveripine; TDF: Tenofovir; Kaletra: Lopinavir+Ritonavir; 3TC: Lamivudine; ddI: Didanosine; Author Manuscript

This article is protected by copyright. All rights reserved Table 2. Demographics, oral hygiene behavior and periodontal characteristics of the groups studied Variable HIV-1-infected Non-HIV-1-infected p value (n = 27) (n = 30) Age 10.5 (3.6), 12 (3 -18) 8.9 (2.1), 9 (5 - 14) 0.059 Gender§ 0.439 Female 15 (55.6) 15 (50.0) Male 12 (44.4) 15 (50.0) Tooth brushing§#φ 0.166 Once/day 4 (18.2) 1 (3.4) 2-3 times/day 14 (63.6) 19 (65.6) >3 times/day 4 (18.2) 9 (31.0) Who does tooth brushing?§#φ 0.207 Him (Her) self 17 (77.3) 26 (89.7) The responsible adult 5 (22.7) 3 (10.3) Dental Floss§#φ None 20 (90.9) 12 (41.4) 0.001 Once/day 2 (9.1) 14 (48.3) Twice/day 0 3 (10.3) Fluoride§#φ 0.724 None 1 (4.5) 2 (6.9) Toothpaste 16 (72.7) 18 (62.1) Toothpaste /Mouthwash 5 (22.7) 9 (31.0) Sugar intake§#φ 0.378 Once/day 7 (31.8) 9 (31.0) 2-3 times/day 7 (31.8) 14 (48.3) >3 times/day 8 (36.4) 6 (20.7) VSB¶ 22.9 (14.5), 22.1 (6.2 – 47.3) 23.2 (25.3), 15.7 (0 – 100.0) 0.346 BPO¶ 7.2 (3.9), 7.1 (0 -12.5) 8.1 (9.3), 6.2 (0 - 45.8) 0.701 §Data presented as N (%), ¶Data presented as mean (SD), median (range), #Data refer to 22 HIV-infected children, φData refer to 29 non-HIV-infected children; BOP (bleeding on probing), SD (Standard deviation); VSB (visible Author Manuscript supragingival biofilm); p value refers to Mann-Whitney U test for continuous variables and chi square test or Fisher exact test for categoric variables.

This article is protected by copyright. All rights reserved Table 3. Estimated OTU richness and diversity indices of partial 16S rRNA gene sequences of bacteria associated with oral samples of HIV-infected and non-HIV-infected children

Sample Chao-1 Shannon

HIV-1-infected Non-HIV-1-infected HIV-1-infected Non-HIV-1-infected Subgingival biofilm 872.71 (±222.44) 588.10 (±216.54) 4.43 (±0.47) 4.41 (±0.53)

Supragingival biofilm 913.83 (±311.94) 555.33 (±161.10) 5.12 (±0.25) 4.00 (±0.61)

Dorsal tongue biofilm 606.00 (±278.57) 694.08 (±336.29) 4.14 (±0.41) 4.15 (±0.96)

Saliva 736.23 (±277.65) 802.07 (±378.92) 4.16 (±0.77) 4.25 (±0.58)

Data presented as mean (±SD) Author Manuscript

Subgingival biofilm Genus (n = 7) (n = 11)

Veillonella 23.4 (14.7), 20.3 (5.7 – 44.3) 11.0 (10.6), 7.81 (1.50 – 31.5) 0.097 0.973

Prevotella 9.97 (11.0), 8.30 (0.60 – 31.9) 5.16 (5.39), 4.36 (0,20 – 16.5) 0.205 0.753

Supragingival biofilm Phylum (n = 6) (n = 11)

Firmicutes 95.4 (3.10), 96.4 (90.6 – 98.4) 41.5 (27.6), 32.5 (15.0 – 95.4) 0.003 2.144

Proteobacteria 0.65 (0.49), 0.44 (0.20 – 1.30) 18.9 (21.9), 6.3 (0.20 – 55.8) 0.027 1.271

Fusobacteria 0.38 (0.40), 0.25 (0.10 – 1.20) 25.2 (24.0), 27.2 (0.30 – 62.4) 0.004 1.999

Genus

Neisseria 0.28 (0.25); 0.17 (0.10 -0.60) 12.2 (14.9), 3.70 (0.10 – 46.1) 0.027 1.271

Streptococcus 77.8 (9.30), 75.8 (68.5 – 88.5) 21.2 (15.8); 15.0 (3.5 – 48.1) 0.001 2.708

Author Manuscript Leptotrichia 0.26 (0.33), 0.19 (0 – 0.90) 16.3 (15.4), 15.3 (0.20 – 38.4) 0.003 2.144

Veillonella 10.0 (18.2), 3.18 (0 – 54.4) 23.8 (19.8), 16.5 (1.50 – 71.3) 0.017 1.266

L Prevotella 3.30 (4.64), 0.45 (0.10 – 11.5) 15.6 (13.1), 13.3 (0.10 – 44.1) 0.021 1.210

Neisseria 20.6 (22.8), 14.8 (1.30 – 67.8) 7.93 (6.52), 8.32 (0.20 – 21.7) 0.280 0.498

Leptotrichia 15.2 (19.5), 6.29 (0.20 – 49.5) 8.49 (7.47), 5.17 (0.10 – 21.5) 0.887 0.069

Saliva Genus (n = 15) (n = 26)

Prevotella 6.83 (12.3), 1.00 (0.20 – 44.5) 14.9 (16.9), 9.10 (0.10 – 61.0) 0.026 0.739

Data presented as mean (SD), median (range); effect size (d): large = 0.80, medium = 0.50 and small = 0.20 Author Manuscript

This article is protected by copyright. All rights reserved Figure legends Figure 1. Phylum - Relative abundances of partial 16S rRNA gene sequences of bacteria associated with oral samples: subgingival biofilm (SB), supragingival biofilm (SP), dorsal tongue biofilm (DT) and saliva (S) of HIV-infected (H) and non-HIV- infected children (N). The most represented phyla were Firmicutes (~58%), Fusobacteria (~14%) and Proteobacteria (~13%).

Figure 2. Genus - Relative abundances of partial 16S rRNA gene sequences of bacteria associated with oral samples: subgingival biofilm (SB), supragingival biofilm (SP), dorsal tongue biofilm (DT) and saliva (S) of HIV-infected (H) and non-HIV-infected children (N). The most dominant genera were Streptococcus (~37%), Veillonella (~12%) and Neisseria (~9%).

Figure 3. Principal component analysis (PCA) of the partial 16S rRNA gene sequences of the bacteria associated with oral samples of HIV-1 infected and non-HIV-1 infected children. (a) subgingival biofilm HIV-1 infected (SBY) and non-HIV-1 infected (SBN); (b) supragingival biofilm HIV-1 infected (SPY) and non-HIV infected (SPN); (c) dorsal tongue biofilm HIV-1 infected (DTY) and non-HIV-1 infected (DTN); (d) saliva HIV-1 infected (SY) and non-HIV-1 infected (SN). Author Manuscript