Journal of Medical Microbiology (2015), 64, 1094–1101
DOI 10.1099/jmm.0.000128
The oral microbiome in human immunodeficiency
virus (HIV)-positive individuals
James O. Kistler,1 Pratanporn Arirachakaran,2 Yong Poovorawan,3
Gunnar Dahlén4 and William G. Wade1
Correspondence
William G. Wade
[email protected]
1
Centre for Immunobiology, Blizard Institute, Barts and The London School of Medicine and
Dentistry, Queen Mary University of London, London, UK
2
Infectious Diseases Clinic, Dental Hospital, Faculty of Dentistry, Chulalongkorn University,
Bangkok, Thailand
3
Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok,
Thailand
4
Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy, University of
Gothenburg, Sweden
Received 25 March 2015
Accepted 7 July 2015
Human immunodeficiency virus (HIV) infection is associated with a range of oral conditions,
and increased numbers of disease-associated microbial species have previously been found
in HIV-positive subjects. The aim of this study was to use next-generation sequencing to
compare the composition of the oral microbiome in HIV-positive and -negative individuals. Plaque
and saliva were collected from 37 HIV-positive individuals and 37 HIV-negative individuals, and
their bacterial composition determined by pyrosequencing of partial 16S rRNA genes. A total of
855 222 sequences were analysed. The number of species-level operational taxonomic units
(OTUs) detected was significantly lower in the saliva of HIV-positive individuals (mean5303.3)
than in that of HIV-negative individuals (mean5365.5) (P,0.0003). Principal coordinates analysis
(PCoA) based on community membership (Jaccard index) and structure (Yue and Clayton
measure of dissimilarity) showed significant separation of plaque and saliva samples [analysis of
molecular variance (AMOVA), P,0.001]. PCoA plots did not show any clear separation based
on HIV status. However, AMOVA indicated that there was a significant difference in the
community membership of saliva between HIV-positive and -negative groups (P50.001). Linear
discriminant analysis effect size revealed an OTU identified as Haemophilus parainfluenzae to be
significantly associated with HIV-positive individuals, whilst Streptococcus mitis/HOT473 was
most significantly associated with HIV-negative individuals. In conclusion, this study has
confirmed that the microbial composition of saliva and plaque is different. The oral microbiomes
of HIV-positive and -negative individuals were found to be similar overall, although there were
minor but significant differences in the composition of the salivary microbiota of the two groups.
INTRODUCTION
There were an estimated 35 million people infected with
the human immunodeficiency virus (HIV) at the end of
Abbreviations: AMOVA, analysis of molecular variance; HAART, highly
active antiretroviral therapy; HIV, human immunodeficiency virus; LDA,
linear discriminant analysis; LEfSe, LDA effect size; OTU, operational
taxonomic units; PCoA, principal coordinates analysis
The NCBI SRA database accession number for the raw sequence data
generated in this study is SRP058922.
Four supplementary tables, one supplementary figure and a supplementary dataset are available with the online Supplementary Material.
1094
2013 and AIDS-related illnesses were responsible for 1.5
million deaths in a single year (Joint United Nations Programme on HIV/AIDS, 2014). Wider and improved
access to highly active antiretroviral therapy (HAART) in
recent years has led to an increase in life expectancy of
HIV-infected individuals (World Health Organization,
2013).
HIV infection is associated with a number of oral conditions linked to lowered CD4+T-cell counts. These
include opportunistic infections by Candida spp. (candidiasis) and by viruses such as herpes simplex virus, varicella-zoster virus, Epstein–Barr virus and human
papilloma viruses (Reznik, 2005). Oral candidiasis is a
Downloaded from www.microbiologyresearch.org by
000128 G 2015 The Authors
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:07:38
Printed in Great Britain
HIV infection and the oral microbiome
particularly common problem in HIV-infected individuals,
although the introduction of HAART has led to a decline in
its incidence (Hodgson et al., 2006). Infection with HIV is
also associated with the periodontal diseases: linear gingival
erythema, nectrotizing ulcerative gingivitis (NUG) and
acute necrotizing periodontitis (ANP) (Mataftsi et al.,
2011). NUG and ANP typically have a sudden onset and
can cause severe pain. ANP is characterized by a rapid
destruction of periodontal tissue, which may result in
tooth loss. Whilst a number of studies have investigated
the bacteria associated with these forms of periodontal disease (Aas et al., 2007; Murray et al., 1989; Paster et al., 2002;
Ramos et al., 2012; Rams et al., 1991), relatively few studies
have compared the normal oral microbiome in
HIV-infected and uninfected individuals. One study used
the Human Oral Microbe Identification Microarray
(HOMIM) to compare the proportions of approximately
300 of the most prevalent oral species on the dorsal surfaces
of the tongues of antiretroviral-treated and untreated HIVinfected individuals, and an uninfected control group
(Dang et al., 2012). The authors detected significantly
increased proportions of pathogenic/disease-associated
species, including Campylobacter concisus, Campylobacter
rectus, Prevotella pallens and Megasphaera micronuciformis,
in the untreated HIV-infected group relative to the uninfected control group (Dang et al., 2012). Another study
using HOMIM, denaturing gradient gel electrophoresis
and culture compared the salivary microbiota in HIV-positive and HIV-negative individuals, and monitored the
changes in microbial diversity after HAART in the HIVpositive group (Li et al., 2014). The authors found that
the microbial diversity of saliva was lower in the HIV-positive group than in the HIV-negative group, and that diversity was reduced following HAART treatment.
A recent study used an open-ended deep sequencing
approach to characterize the composition of both the
oral mycobiome and bacteriome in 12 HIV-infected and
12 uninfected individuals (Mukherjee et al., 2014). The
authors reported no significant differences in the bacterial
community composition of oral rinse samples from these
groups. However, they revealed differences in the fungal
communities (mycobiome). In particular, Pichia species
were associated with uninfected individuals and had a
negative correlation to the presence of Candida species.
The authors also showed that Pichia spp. could inhibit
Candida growth in vitro. Whilst the authors reported that
the bacteriomes of HIV-infected and uninfected individuals were similar, it is possible that differences in specific
habitats such as tooth surfaces were overlooked, as a
single oral rinse sample was used for each individual.
In addition, the number of participants in each group
was relatively low.
The aim of this study was to use next-generation sequencing to compare the composition of oral bacterial communities present in plaque and saliva of HIV-positive and
-negative individuals.
http://jmm.sgmjournals.org
METHODS
Study groups. Ethical approval for this study was granted by the
Ethics Committee of the Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand, and written informed consent was
obtained for all of the participants. A group of HIV-positive patients
was recruited for cross-sectional comparison to a group of HIVnegative subjects (Tables 1 and S1, available in the online Supplementary Material). The groups were matched for age and gender
and all of the subjects were non-smokers who had not used antibiotics
during the 6 months prior to sampling. All of the individuals were
Thai who lived in Bangkok and visited Chulalongkorn University
dental clinics for dental care. The subjects did not have any recent
history of oral lesions (6 months prior to sampling), oral mucositis,
active caries involvement or dental implants. HIV-positive subjects
had been infected for at least 10 years prior to sampling and had been
undergoing antiretroviral therapy for at least 5 years. The HIV viral
load of the HIV-seropositive patients was determined using quantitative real-time PCR (Roche Molecular Systems) and expressed as the
number of viral copies ml21. The number of CD4+ cells of the HIVpositive subjects was determined by flow cytometry with a Becton
Dickinson instrument and expressed as cells mm23.
Sample collection. Dental plaque and saliva were collected from
both groups. Probing for pocket depth and bleeding was performed
after saliva collection, but before plaque collection, during the same
visit. Plaque samples were taken using a sterile curette from the buccal
surfaces of three teeth (one molar, one premolar and one incisor) in
each quadrant by scraping along the tooth in the gingival sulcus and
pooled into 0.5 ml of sterile Tris/EDTA buffer. No sites with deep
pockets (w3 mm) were included in the sampling. If a deep pocket
was located at a sampling tooth/site, the adjacent tooth was used.
There was no evidence of bleeding on probing at the sites used for
sampling plaque. Two millilitres of saliva was collected by expectoration into 10 ml sterile tubes. Plaque and saliva samples were
stored at 270 uC and processed within 24 h of collection.
DNA extraction. DNA was extracted from saliva and plaque using
the GenElute Bacterial DNA Extraction kit (Sigma-Aldrich), with an
additional lysis step to increase the recovery of Gram-positive bacterial DNA: the samples were incubated with a 45 mg lysozyme ml21
solution at 37 uC for 30 min.
16S rRNA gene PCR and 454 pyrosequencing. The protocol used
was as described previously (Kistler et al., 2013) with some minor
modifications. PCR amplification of a fragment of the 16S rRNA
gene, approximately 500 bp in length covering the V1–V3 hypervariable regions, was performed using composite fusion primers. The
fusion primers comprised the broad-range 16S rRNA gene primers 27
Table 1. Summary of the study groups
No. of subjects
Mean (¡SD ) age (years)
Male : female
Mean CD4+T-cell count (¡SD )
(cells mm23)
NA ,
HIV-positive
HIV-negative
37
37
44 (¡10)
18 : 19
510 (¡232)
46 (¡10)
17 : 20
NA
Not applicable.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:07:38
1095
J. O. Kistler and others
FYM (Frank et al., 2008) and 519 R (Lane et al., 1985) along with
Roche GS-FLX Titanium Series adaptor sequences (A and B) for 454
pyrosequencing using the Lib-L emulsion-PCR method. Previously
described 12-base error-correcting ‘Golay’ barcode sequences (Fierer
et al., 2008) were incorporated into the forward primers to enable
multiplexing of samples in the same sequencing run. The appropriate
barcoded forward primer and reverse primer were used in PCRs along
with Extensor High-Fidelity PCR Mastermix (Thermo-Scientific).
The PCR conditions were as follows: 5 min initial denaturation step at
95 uC, followed by 25 cycles of 95 uC for 45 s, 53 uC for 45 s and 72 uC
for 45 s and a final extension of 72 uC for 5 min. PCR amplicons were
then purified using the QIAquick PCR purification kit (Qiagen)
according to the manufacturer’s instructions. The size and purity of
the amplicons were checked using the Agilent DNA 1000 kit and the
Agilent 2100 Bioanalyser. Quantification of the amplicons was performed by means of a fluorometric assay using the Quant-iT Picogreen fluorescent nucleic acid stain (Invitrogen). The amplicons were
then pooled together at equimolar concentrations (1|109 molecules ml21). Emulsion-PCR and unidirectional sequencing of the
samples were performed using the Lib-L kit and the Roche 454 GSFLX+Titanium series sequencer by the Department of Biochemistry,
Cambridge University, Cambridge, UK.
Statistical analysis. Analysis of molecular variance (AMOVA)
(Excoffier et al., 1992), as implemented by mothur, was used to
determine if there were any statistically significant differences between
groups and sample types in the distance matrices based on the Jaccard
and thetaYC indices. Linear discriminant analysis (LDA) effect size
(LEfSe) (Segata et al., 2011) was used to identify OTUs that were
significantly differentially abundant between saliva and plaque and
between HIV-positive and -negative groups. The alpha values for the
factorial Kruskal–Wallis and pairwise Wilcoxon tests were set to 0.05
and the LDA score threshold for discriminative features was set to 3.5.
Two sample t-tests were performed in R to determine if there were
any significant differences in OTU richness and diversity between
HIV-positive and -negative groups. Correlations between the relative
abundances of OTUs and metadata for the HIV-positive group,
including CD4+ cell counts and subject age, were determined using
Pearson’s rank correlation coefficient as implemented by mothur in
the ‘otu.association’ command; OTUs that were detected in less than
50 % of the saliva or plaque samples were not included in the analysis.
RESULTS
Pyrosequencing
Sequence analysis. Sequence analysis was performed using the
‘mothur’ software suite version 1.33 (Schloss et al., 2009), following
the 454 standard operating procedure (Schloss & Westcott, 2011) on
http://www.mothur.org. The sequences were denoised using the
AmpliconNoise algorithm, as implemented by mothur. Sequences
that were less than 440 bases in length and/or had one of the following: w2 mismatches to the primer, w1 mismatch to the barcode
regions, and homopolymers of w8 bases in length, were discarded.
The remaining sequences were trimmed to remove primers and
barcodes and aligned to the SILVA 16S rRNA gene reference alignment. The UChime algorithm was used to identify chimeric
sequences, which were then removed from the dataset. Sequences
were clustered into operational taxonomic units (OTUs) at a genetic
distance of 0.015 using the average neighbour algorithm and identified using a naive Bayesian classifier with the Human Oral Microbiome Database (HOMD) reference set (version 13). A distance of
0.015 was chosen because many named oral bacterial species have
high sequence identity in their 16S rRNA genes, particularly among
the streptococci, Actinomyces and Neisseria. Therefore, a number of
different species are likely to be combined into the same OTU when
applying the more commonly used distance of 0.03. In addition,
species-level phylotypes in the HOMD have been defined based on a
98.5 % 16S rRNA gene sequence identity cut-off (Dewhirst et al.,
2010). For those OTUs that could not be identified using the Bayesian
classifier, representative sequences were obtained in mothur (the
sequence with the smallest distance to all other sequences in that
OTU) and identified using BLAST against the HOMD reference set.
The possible alternatives for the species identification were then
provided. In order to perform OTU-based comparisons of bacterial
diversity, the number of sequences in each sample was normalized by
subsampling down to the number of sequences equal to that of the
sample with the lowest number. The diversity of the communities was
estimated and compared in mothur using Simpson’s inverse diversity
index (Simpson, 1949). Good’s non-parametric coverage estimator
was used to determine how well sampled the communities were
(Good, 1953). The beta-diversity of the samples was analysed using
distance matrices generated using the Jaccard index for community
membership and the Yue and Clayton measure of dissimilarity
(thetaYC calculator) for community structure (Yue & Clayton, 2005).
The distance matrices were visualized using dendrograms and principal coordinates analysis (PCoA) plots. Three-dimensional PCoA
plots were generated in R (http://www.r-project.org) using the ‘rgl’
package.
1096
A total of 855 222 sequences were included in the final
analysis after filtering and removal of chimeric sequences.
This resulted in a mean of 5980 per sample (range: 3583–
9464). Five samples were not included in the pyrosequencing run due to poor PCR amplification. This resulted in
a final total of 36 plaque and 36 saliva samples in the
HIV-positive group and 35 plaque and 36 saliva samples
in the HIV-negative group for comparison. The mean
numbers of quality-filtered sequences per sample were
6355 (range: 3587–9245) and 5599 (range: 3583–9464) in
the HIV-positive and -negative groups respectively.
Composition of plaque and saliva samples
The sequences were assigned to a total of 12 phyla. The predominant phyla in both plaque and saliva were Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and
Proteobacteria. Other phyla detected, but not in every
sample, included Chloroflexi, GN02, SR1, Spirochaetes,
Synergistetes, TM7 and Tenericutes. The relative abundances
of the predominant phyla in plaque and saliva are shown in
Fig. S1. A total of 168 different genera were detected among
all of the samples. The predominant genera in plaque (constituting w5 % mean relative abundance) were Streptococcus,
Corynebacterium,
Prevotella,
Capnocytophaga,
Actinomyces, Veillonella and Selenomonas, whilst in saliva
the predominant genera were Streptococcus, Prevotella, Veillonella and Rothia. The bacterial composition of all the
samples (classified to species level where possible) is
shown in Dataset S1.
Comparisons of OTU richness and diversity
Table S2 shows the OTU richness and diversity (Simpson’s
inverse diversity index) of each of the plaque and saliva
samples included in the analysis (when subsampled to
3583 sequences per sample). The mean number of OTUs
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:07:38
Journal of Medical Microbiology 64
HIV infection and the oral microbiome
detected in the saliva of the HIV-positive individuals was
significantly lower than in the HIV-negative individuals
(two sample t-test, Pv0.0003) (Fig. 1a). Two saliva
samples from HIV-positive individuals had a particularly
reduced richness with 180 and 184 OTUs detected in
each sample. Interestingly, the same individuals had the
lowest richness in their plaque samples (141 and 177
OTUs). There was, however, no significant difference
(P50.058) between the mean number of OTUs detected
in the plaque samples of the HIV-positive and -negative
groups (Fig. 1a). In addition, there were no significant
differences in the Simpson’s inverse diversity index of
saliva or plaque samples of the two groups. The mean
(a)
450
400
350
*
Number of OTUs
300
250
200
150
100
50
0
Plaque_HIV_neg Plaque_HIV_pos
Saliva_HIV_neg
Saliva_HIV_pos
(b)
40
Simpson's inverse diversity index
35
30
25
20
15
diversity of plaque samples was higher than that of saliva
samples (Fig. 1b).
Comparisons of bacterial community membership
and structure
The community membership and structure of plaque and
saliva samples from HIV-positive and -negative groups
was compared using PCoA plots. Comparisons of samples
from both HIV-positive and negative groups in PCoA plots
revealed clear separation of plaque and saliva (Fig. 2).
AMOVA tests confirmed that saliva and plaque were
highly significantly different (Pv0.001 for both membership and structure). The plots did not show a clear separation/difference between the HIV-positive and -negative
groups for either the plaque or saliva samples (Fig. 3).
AMOVA confirmed that there was no significant difference
in community structure between the groups. However,
AMOVA indicated that there was a significant difference
between the community membership of saliva, but not
plaque, of HIV-negative and -positive subjects (P50.001)
(Fig. 4).
Comparison using LEfSe revealed 121 OTUs that were significantly differentially abundant between saliva and plaque
samples. The differentially abundant OTUs with LDA
scores of i3.5 are shown in Fig. 5. The OTU most significantly associated with saliva was identified as Streptococcus
salivarius/Streptococcus vestibularis, whilst a Corynebacterium matruchotii OTU was most significantly associated
with plaque. Comparison of the saliva of HIV-positive subjects to that of HIV-negative subjects revealed 116 OTUs
that were significantly differentially abundant. However,
only three OTUs had LDA scores of i3.5 and many of
the associated OTUs were detected at low relative abundances. Of the OTUs with LDA scores of i3.5, a Haemophilus parainfluenzae OTU was associated with HIVpositive individuals, whilst a Streptococcus mitis/Streptococcus pneumoniae/HOT473 OTU and a Streptococcus mitis
bv.2 OTU were associated with HIV-negative individuals.
LEfSe comparison of plaque from HIV-positive and -negative individuals was not performed, as there was no significant difference in community membership or structure by
AMOVA.
10
Correlation of OTUs with CD41 cell counts and
subject age
5
0
Plaque_HIV_neg Plaque_HIV_pos
Saliva_HIV_neg
Saliva_HIV_pos
Fig. 1. Bar charts comparing the mean species-level OTU richness (a) and Simpson’s inverse diversity index (b) in plaque and
saliva samples of the HIV-positive and -negative groups. The saliva of HIV-positive individuals had a significantly lower richness
than the saliva of HIV-negative individuals (indicated by the asterisk) using a two-sample t-test. There was no significant difference
in Simpson’s diversity index between groups. The error bars indicate SEM .
http://jmm.sgmjournals.org
The OTUs that showed a statistically significant correlation
(Pearson’s rank correlation coefficient) with CD4+T-cell
counts and with subject age in the HIV-positive group
are shown in Tables S3 and S4. For both plaque and
saliva samples, stronger correlations were obtained with
age than with the CD4+ cell counts. The OTUs that
showed the strongest negative correlations with CD4+
cell counts were OTU 30 Alloprevotella tannerae (20.42,
Pv0.009) and OTU 373 Eubacterium yurii (20.45,
Pv0.004) in saliva and plaque, respectively. The OTUs
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:07:38
1097
J. O. Kistler and others
(a)
(b)
–0.1
–0.2
0.2 –0.2
0.1
0
–0.1
0
0.1
0.2
0.3
–0.2
0.2
0.4
0.2
0
–0.4
–0.2
0
0.2
0.4
0.2
0.1
0
0
–0.2
–0.1
PC3
PC3
–0.4
–0.2
PC2
PC2
PC1
PC1
Fig. 2. PCoA plots comparing plaque and saliva from HIV-positive and -negative individuals based on their community membership (Jaccard index) (a) and structure (thetaYC calculator) (b). Plaque samples are shown in red and saliva samples are in
blue. Plaque and saliva had a highly significantly different community membership and structure by AMOVA (P,0.001 for
both). The proportion of variance shown for (a) and (b), respectively, was: PC156.1 % and 18.4 %, PC252.4 % and 7.3 %,
PC351.5 % and 5.5 %.
(a)
(b)
0.2 –0.2
0.1
0
0.1
–0.2
–0.1
0
0.1
0.2
0.3
0.4
0.2
–0.2 0
–0.4
–0.2
0
0.2
0.4
0.2
0.1
0.2
0
0
–0.1
PC3
–0.2
PC3
–0.2
–0.4
PC2
PC2
PC1
PC1
Fig. 3. PCoA plots comparing plaque and saliva from HIV-positive and -negative individuals based on their community membership (Jaccard index) (a) and structure (thetaYC calculator) (b). Red, saliva from HIV-positive subjects; blue, saliva from
HIV-negative subjects; purple, plaque from HIV-positive subjects; green, plaque from HIV-negative subjects. There was a significant difference between the community membership, but not structure, of saliva between groups using AMOVA
(P50.001). No other significant differences were detected. The proportion of variance shown for (a) and (b), respectively,
was: PC156.1 % and 18.4 %, PC252.4 % and 7.3 %, PC351.5 % and 5.5 %.
1098
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:07:38
Journal of Medical Microbiology 64
HIV infection and the oral microbiome
0.2
0.1
PC2
0
–0.1
–0.2
–0.3
0.15
0.1
0.05
0
–0.05
–0.1
–0.15
0.2
0.1
0
PC3
PC1
Fig. 4. PCoA plot comparing saliva from HIV-positive and
-negative individuals based on their community membership (Jaccard index). Red, saliva from HIV-positive subjects; blue, saliva
from HIV-negative subjects. There was a significant difference in
community membership between the two groups by AMOVA
(P50.001). The proportion of variance shown is: PC156.1 %,
PC252.4 %, PC351.5 %.
most strongly positively correlated with CD4+T-cell counts
were OTU 623 H. parainfluenzae (0.46, Pv0.004) and
OTU 269 Actinomyces sp. (0.50, Pv0.002) in saliva and
plaque, respectively.
DISCUSSION
This study has comprehensively characterized the bacterial
composition of dental plaque and saliva of HIV-positive
individuals, at the species level, and compared it with
that of HIV-negative individuals. The results confirmed
the findings of previous studies, which showed that saliva
and plaque have a different bacterial composition (Segata
et al., 2012; Simón-Soro et al., 2013). In addition, the
results showed that plaque had a higher bacterial diversity
(Simpson’s inverse diversity index) than saliva. Species that
were most strongly associated with saliva included salivarius- and mitis-group streptococci, Rothia mucilaginosa and
Prevotella melaninogenica whilst species most strongly
associated with plaque included Corynebacterium matruchotii, Streptococcus sp. HOT058 and Capnocytophaga granulosa. The hard, non-shedding tooth surfaces on which
plaque forms provide a distinct microbial habitat to the
shedding mucosal surfaces of the mouth and is thus associated with a differing microbial community structure
(Mager et al., 2003; Segata et al., 2012). Using chequerboard
http://jmm.sgmjournals.org
DNA–DNA hybridization, Mager et al. (2003) showed that
the distribution of 40 selected bacterial species in saliva was
most similar to that found on the tongue surfaces,
suggesting that saliva predominantly comprises species
that are shed from tongue biofilms. In that study, saliva
and tongue communities were characterized by increased
numbers of Prevotella melaninogenica and Veillonella
parvula relative to other oral sites/samples. Nextgeneration sequencing data from the Human Microbiome
Project also showed that saliva samples were highly similar
in composition to samples from the tongue dorsum (Segata
et al., 2012). Supra- and subgingival plaque had a distinct
composition from samples from other oral sites and was
characterized by having significantly higher proportions
of members of the orders Burkholderiales, Cardiobacteriales,
Flavobacteriales, Fusobacteriales, Neisseriales and Pasteurellales. However, the sequences were not classified to the
species level.
Comparisons of the community composition of plaque and
saliva of HIV-positive and HIV-negative individuals in
PCoA plots showed that their bacterial communities were
largely similar, supporting the findings of a recent deepsequencing study (Mukherjee et al., 2014). The plaque
samples in the present study were, however, only taken
from periodontally healthy sites in both groups. It is therefore possible that there are significant differences in plaque
composition between HIV-infected and uninfected individuals at inflamed/diseased sites. Differences in OTU
community membership between groups were detected in
saliva by AMOVA and a number of species-level OTUs
were identified as being significantly differentially abundant. In addition, the saliva of the HIV-positive individuals
had a significantly lower OTU richness than that of the
HIV-negative individuals. This is in agreement with the
findings of a previous study that compared the salivary
microbiota of HIV-positive and -negative individuals
using culture and other culture-independent methods (Li
et al., 2014). The authors reported a decreased microbial
diversity (Shannon diversity index) in the saliva of HIVpositive subjects compared with that of the negative control group and also showed that the microbial diversity
within the HIV-positive group was reduced following
HAART. There is evidence that HAART can reduce salivary
flow (Navazesh et al., 2009) and this in turn might affect
the richness and composition of the salivary microbiota.
A previous study has shown that individuals with reduced
salivary secretion have increased numbers of lactobacilli,
which are caries-associated, in their saliva compared with
a control group (Almståhl & Wikström, 1999). All of the
HIV-positive patients in the present study were undergoing
HAART. The importance of the OTUs that were associated
with HIV-positive individuals is unclear as they were found
to be relatively common oral commensal species.
An H. parainfluenzae OTU was most significantly associated with the HIV-positive group. This commensal species
is a member of the HACEK group of Gram-negative bacilli
that have been implicated in various opportunistic
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:07:38
1099
J. O. Kistler and others
OTU2_Streptococcus_salivarius_vestibularis
OTU3_Streptococcus_mitis_pneumoniae_HOT423_HOT064
OTU7_Rothia_mucilaginosa
OTU9_Streptococcus_infantis_HOT065
OTU4_Prevotella_melaninogenica
OTU10_Haemophilus_parainfluenzae
OTU16_Gemella_sanguinis
OTU19_Streptococcus_mitis_HOT431
OTU17_Streptococcus_parasanguinis
OTU13_Neisseria_flavescens_subflava
OTU15_Granulicatella_adiacens
OTU35_Prevotella_histicola
OTU41_Prevotella_sp_HOT306
OTU22_Gemella_morbillorum_haemolysans
OTU44_Actinomyces_odontolyticus_HOT180
OTU47_Streptococcus_mitis_bv2
OTU34_Prevotella_pallens
OTU30_Alloprevotella_tannerae
OTU52_Bacteroidales_G2_sp_HOT274
OTU42_Ottowia_sp_HOT894
OTU28_Cardiobacterium_hominis
OTU36_Cardiobacterium_valvulum
OTU31_Actinomyces_sp_HOT448
OTU27_Capnocytophaga_leadbetteri
OTU26_Campylobacter_gracilis
OTU24_Dialister_invisus
OTU25_Tannerella_sp_HOT286
OTU12_Streptococcus_cristatus
OTU21_Capnocytophaga_granulosa
OTU11_Streptococcus_sp_HOT058
OTU6_Corynebacterium_matruchotii
OTU5_Corynebacterium_matruchotii
–6.0
–4.8
–3.6
–2.4
–1.2
0.0
1.2
2.4
3.6
4.8
6.0
LDA score (log10)
Plaque
Saliva
Fig. 5. Significantly differentially abundant OTUs between saliva and plaque using LEfSe. A total of 121 OTUs were significantly differentially abundant between saliva and plaque. The 32 OTUs shown are those with effect sizes of ¢3.5.
infections including endocardial infections, upper respiratory tract infections, brain abscesses and joint infections (Janda,
2013). Whilst H. parainfluenzae was associated with the HIVpositive individuals, within the HIV-positive group it was positively correlated with CD4+ cell counts. This suggests that H.
parainfluenzae does not increase in proportion as the disease
progresses and CD4+ cell numbers decline.
In conclusion, this study found that the oral microbiomes
of HIV-positive and -negative individuals were similar
overall, although minor but significant differences in the
composition of the salivary microbiota were detected.
The results also confirmed that plaque and saliva have a
distinct microbial composition.
1100
ACKNOWLEDGEMENTS
This study was supported by the Higher Education Research Promotion
and National Research University Project of Thailand, Office of the
Higher Education Commission (HR1155A). This research utilized
Queen Mary’s MidPlus computational facilities, supported by QMUL
Research-IT and funded by EPSRC grant EP/K000128/1.
REFERENCES
Aas, J. A., Barbuto, S. M., Alpagot, T., Olsen, I., Dewhirst, F. E. &
Paster, B. J. (2007). Subgingival plaque microbiota in HIV positive
patients. J Clin Periodontol 34, 189–195.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:07:38
Journal of Medical Microbiology 64
HIV infection and the oral microbiome
Almståhl, A. & Wikström, M. (1999). Oral microflora in subjects with
reduced salivary secretion. J Dent Res 78, 1410–1416.
patients: identification of Pichia as an antagonist of opportunistic
fungi. PLoS Pathog 10, e1003996.
Dang, A. T., Cotton, S., Sankaran-Walters, S., Li, C.-S., Lee, C.-Y. M.,
Dandekar, S., Paster, B. J. & George, M. D. (2012). Evidence of an
Murray, P. A., Grassi, M. & Winkler, J. R. (1989). The microbiology of
increased pathogenic footprint in the lingual microbiome of
untreated HIV infected patients. BMC Microbiol 12, 153.
Dewhirst, F. E., Chen, T., Izard, J., Paster, B. J., Tanner, A. C., Yu,
W. H., Lakshmanan, A. & Wade, W. G. (2010). The human oral
HIV-associated periodontal lesions. J Clin Periodontol 16, 636–642.
Navazesh, M., Mulligan, R., Karim, R., Mack, W. J., Ram, S., Seirawan,
H., Greenspan, J., Greenspan, D., Phelan, J., Alves, M. & Oral
Substudy of the Women’s Interagency HIV Study Collaborative
Study Group* (2009). Effect of HAART on salivary gland function
microbiome. J Bacteriol 192, 5002–5017.
in the Women’s Interagency HIV Study (WIHS). Oral Dis 15, 52–60.
Excoffier, L., Smouse, P. E. & Quattro, J. M. (1992). Analysis of
Paster, B. J., Russell, M. K., Alpagot, T., Lee, A. M., Boches, S. K.,
Galvin, J. L. & Dewhirst, F. E. (2002). Bacterial diversity in
molecular variance inferred from metric distances among DNA
haplotypes: application to human mitochondrial DNA restriction
data. Genetics 131, 479–491.
Fierer, N., Hamady, M., Lauber, C. L. & Knight, R. (2008). The
influence of sex, handedness, and washing on the diversity of hand
surface bacteria. Proc Natl Acad Sci U S A 105, 17994–17999.
necrotizing ulcerative periodontitis in HIV-positive subjects. Ann
Periodontol 7, 8–16.
Ramos, M. P., de A., Ferreira, S. M. S., Silva-Boghossian, C. M.,
Souto, R., Colombo, A. P., Noce, C. W. & Gonçalves, L. S. (2012).
Frank, J. A., Reich, C. I., Sharma, S., Weisbaum, J. S., Wilson, B. A. &
Olsen, G. J. (2008). Critical evaluation of two primers commonly
Necrotizing periodontal diseases in HIV-infected Brazilian patients:
a clinical and microbiologic descriptive study. Quintessence Int 43,
71–82.
used for amplification of bacterial 16S rRNA genes. Appl Environ
Microbiol 74, 2461–2470.
Rams, T. E., Andriolo, M. Jr, Feik, D., Abel, S. N., McGivern, T. M. &
Slots, J. (1991). Microbiological study of HIV-related periodontitis.
Good, I. J. (1953). The population frequencies of species and the
estimation of population parameters. Biometrika 40, 237–264.
Hodgson, T. A., Greenspan, D. & Greenspan, J. S. (2006). Oral
lesions of HIV disease and HAART in industrialized countries. Adv
Dent Res 19, 57–62.
Janda, W. M. (2013). Update on the HACEK group of fastidious gram-
negative bacilli, Part I. Clin Microbiol Newsl 35, 87–92.
Joint United Nations Programme on HIV/AIDS (UNAIDS) (2014).
The Gap Report. Geneva: UNAIDS.
Kistler, J. O., Booth, V., Bradshaw, D. J. & Wade, W. G. (2013).
Bacterial community development in experimental gingivitis. PLoS
One 8, e71227.
Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. & Pace,
N. R. (1985). Rapid determination of 16S ribosomal RNA sequences
for phylogenetic analyses. Proc Natl Acad Sci U S A 82, 6955–6959.
Li, Y., Saxena, D., Chen, Z., Liu, G., Abrams, W. R., Phelan, J. A.,
Norman, R. G., Fisch, G. S., Corby, P. M. & other authors (2014).
HIV infection and microbial diversity in saliva. J Clin Microbiol 52,
1400–1411.
J Periodontol 62, 74–81.
Reznik, D. A. (2005). Oral manifestations of HIV disease. Top HIV
Med 13, 143–148.
Schloss, P. D. & Westcott, S. L. (2011). Assessing and improving
methods used in operational taxonomic unit-based approaches for
16S rRNA gene sequence analysis. Appl Environ Microbiol 77,
3219–3226.
Schloss, P. D., Westcott, S. L., Ryabin, T., Hall, J. R., Hartmann, M.,
Hollister, E. B., Lesniewski, R. A., Oakley, B. B., Parks, D. H. &
other authors (2009). Introducing mothur: open-source, platform-
independent, community-supported software for describing and
comparing microbial communities. Appl Environ Microbiol 75,
7537–7541.
Segata, N., Izard, J., Waldron, L., Gevers, D., Miropolsky, L., Garrett,
W. S. & Huttenhower, C. (2011). Metagenomic biomarker discovery
and explanation. Genome Biol 12, R60.
Segata, N., Haake, S. K., Mannon, P., Lemon, K. P., Waldron, L.,
Gevers, D., Huttenhower, C. & Izard, J. (2012). Composition of the
adult digestive tract bacterial microbiome based on seven mouth
surfaces, tonsils, throat and stool samples. Genome Biol 13, R42.
Mager, D. L., Ximenez-Fyvie, L. A., Haffajee, A. D. & Socransky, S. S.
(2003). Distribution of selected bacterial species on intraoral surfaces.
Simpson, E. H. (1949). Measurement of diversity. Nature 163, 688.
Mataftsi, M., Skoura, L. & Sakellari, D. (2011). HIV infection and
92, 616–621.
periodontal diseases: an overview of the post-HAART era. Oral Dis
17, 13–25.
World Health Organization (2013). Global Update on HIV Treatment
Mukherjee, P. K., Chandra, J., Retuerto, M., Sikaroodi, M., Brown,
R. E., Jurevic, R., Salata, R. A., Lederman, M. M., Gillevet, P. M. &
Ghannoum, M. A. (2014). Oral mycobiome analysis of HIV-infected
Yue, J. C. & Clayton, M. K. (2005). A similarity measure based on
J Clin Periodontol 30, 644–654.
http://jmm.sgmjournals.org
Simón-Soro, A., Tomás, I., Cabrera-Rubio, R., Catalan, M. D., Nyvad,
B. & Mira, A. (2013). Microbial geography of the oral cavity. J Dent Res
2013: Results, Impact and Opportunities. Geneva: World Health
Organization.
species proportions. Commun Stat Theory Methods 34, 2123–2131.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 21:07:38
1101