Table Of ContentJCM Accepts, published online ahead of print on 12 February 2014
J. Clin. Microbiol. doi:10.1128/JCM.02954-13
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1 HIV Infection and Microbial Diversity in Saliva
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3 Yihong Li,a# Deepak Saxena,a Zhou Chen,a Gaoxia Liu,a* Willam R. Abrams,a Joan A.
4 Phelan,b Robert G. Norman,c Gene S. Fisch,c Patricia M. Corby,d Floyd Dewhirst,e
5 Bruce J. Paster,e Alexis S. Kokaras,e Daniel Malamuda
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7 Department of Basic Science and Craniofacial Biology, New York University College of D
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8 Dentistry a; Department of Oral and Maxillofacial Pathology, Radiology, and Medicine, New w
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9 York University College of Dentistry b; Department of Epidemiology and Health Promotion, lo
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10 New York University College of Dentistry c; Bluestone Center for Clinical Research and e
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11 Department of Periodontology and Implant Dentistry, New York University College of Dentistry, f
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12 New York, New York, USAd; The Forsyth Institute, Cambridge, MA and Harvard School of m
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13 Dental Medicine, Boston, Massachusetts, USAe t
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14 //
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16 Running Head: HIV Infection and Microbial Diversity in Saliva s
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19 #Address correspondence to Yihong Li, [email protected] n
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20 Department of Basic Science and Craniofacial Biology, New York University College of p
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21 Dentistry, 345 E. 24th Street, New York, NY 10010, USA il 3
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22 Tel: (212) 998-9607; Fax: (212) 995-4087
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24 *Present address: Gaoxia Liu, Department of Stomatology, Union Hospital, Tongji Medical y
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25 College, Huazhong University of Science and Technology, Wuhan, Hubei, P.R. China 430022 u
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26 Email: [email protected] t
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28 Key words: Microbiology, immunology, retrovirus therapy, saliva, microbial diversity,
29 denaturing gradient gel electrophoresis (DGGE), human oral microbe identification microarray
30 (HOMIM)
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31 ABSTRACT
32 Limited information is available about the effect of human immunodeficiency virus (HIV) and
33 subsequent antiretroviral treatment on host-microbe interaction. This study aimed to determine
34 the salivary microbial composition in 10 HIV-seropositive subjects, before and 6 months after
35 highly active antiretroviral therapy (HAART), compared with that of 10 HIV-seronegative
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36 subjects. Both a conventional culture and two culture-independent analyses were used and
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37 consistently demonstrated differences in microbial composition among the three sets of samples. lo
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38 HIV+ subjects had higher levels of total cultivable microbes, including oral streptococci, ed
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39 lactobacilli, S. mutans, and Candida, in saliva as compared to HIV- subjects. The total cultivable om
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40 microbial level was significantly correlated with CD4+ T cell counts. Denaturing gradient gel t
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41 electrophoresis (DGGE), which compared the overall microbial profiles, showed distinct jc
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42 fingerprinting profiles for each group. Human oral microbe identification microarray (HOMIM), a
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43 which compared the 16S rRNA genes, showed a clear separation among the three sample groups. .o
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44 Veillonella, Synergistetes, and Streptococcus, were present in all 30 saliva samples. Only minor o
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45 changes or no changes were observed in the prevalence of Neisseria, Haemophilus, Gemella,
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46 Leptotrichia, Solobacterium, Parvimonas and RothiaI. Severn genera were detected only in il 3
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47 HIV- samples, including Capnocytophaga, Slackia, Porphyromonas, Kingella, 0
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48 Peptostreptococcaceae, Lactobacillus, and Atopobium. The prevalence of Fusobacterium, b
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49 Campylobacter, Prevotella, Capnocytophaga, Selenomonas, Actinomyces, and Atopobium was u
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50 increased after therapy with HAART. In contrast, the prevalence of Aggregatibacter was
51 significantly decreased after HAART. Findings of this study suggest that HIV infection and
52 therapy with HAART could have a significant effect on salivary microbial colonization and
53 composition.
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55 INTRODUCTION
56 As of 2012, more than 35 million people were living with human immunodeficiency
57 virus (HIV), and more people than ever received life-saving antiretroviral therapy worldwide (1).
58 The availability of antiretroviral therapy has significantly reduced the number of AIDS-related
59 deaths. Concurrently, people living with HIV are continuously challenged by diseases associated
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60 with a compromised host immune system, including opportunistic infections (2). In the oral
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61 cavity, oropharyngeal candidiasis is the most common oral infection (3), and it can be detected in lo
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62 the early stages of HIV infection (4). This opportunistic infection and others could be a d
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63 consequence of immune impairment induced by HIV, changes in saliva composition and m
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64 function (5, 6), the presence of advanced caries lesions (7), and progressive periodontal t
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65 infections (8). jc
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66 Despite the overall decline in HIV-related deaths, studies have suggested a clinical a
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67 association between HIV infection and both caries and periodontal diseases; for example, .o
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68 immunocompromised individuals, especially children and young adult populations, have shown o
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69 an increased prevalence of dental caries (7, 9-11) and necrotizing periodontal diseases (12, 13).
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70 However, a few studies have shown no difference between HIV-infected and healthy subjects in il 3
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71 caries severity, chronic periodontitis, or advanced periodontal diseases (14, 15). Previously, we 0
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72 reported salivary microbial changes in HIV-infected patients (16). Others have observed b
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73 positive correlations between HIV infection and increased colonization of oral Candida (6, 17- u
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74 19). It has also been suggested that impairment of systemic defense mechanisms by reduction of
75 CD4+ T cells below protective levels and impairment of local immunity by reduction of salivary
76 IgA, defensins or epithelial cell-mediated cytokines in the saliva might lead to the conversion of
77 commensal Candida to a microorganism with increased pathogenicity, causing, in turn, an
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78 imbalance in the normal host oral microbial composition and, hence, increased risk for
79 opportunistic infections (20, 21). A dramatic reduction in oral candidiasis after therapy with
80 HAART has been consistently evidenced (22-25); however, the mechanisms underlying host-
81 microbe interactions relative to HIV infection and subsequent HAART, all in the context of the
82 oral microbial composition, are not well understood. Currently, a wide range of molecular
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83 techniques are available to help identify and characterize microorganisms, including sequencing
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84 the genes encoding 16S rRNA, using DNA hybridization with custom designed oligonucleotide lo
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85 probes, fingerprinting the microbial flora with denaturing gradient gel electrophoresis (DGGE), d
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86 and other techniques based on polymerase chain reaction (PCR). m
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87 The present study aimed to evaluate the microbial colonization and composition in t
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88 samples collected from HIV+ subjects and healthy controls. We hypothesized that individuals /jc
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89 immunocompromised by HIV infection were at a higher risk for an increased microbial a
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90 colonization and diversity than healthy controls, leading, in turn, to increased prevalence and .o
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91 severity of oral diseases. We also hypothesized that therapy with HAART could reverse the o
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92 HIV-associated changes in oral microbial composition in saliva, restoring balance in the oral
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93 microbiota and, hence, improving the oral health of individuals with HIV. A conventional culture il 3
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94 method was used to evaluate total cultivable microbes in saliva, and two culture-independent 0
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95 methods based on 16S rRNA were used to determine the effect of HIV infection and HAART on b
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96 changes in salivary microbial composition. The molecular fingerprints generated by DGGE u
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97 provided a direct cross-sectional comparison of microbial composition of the targeted bacterial
98 16S gene (26, 27). In addition, the human oral microbe identification microarray (HOMIM)
99 assay enabled us to further distinguish the observed differences in microbial diversity at
100 microbial genus or species level.
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101 MATERIALS AND METHODS
102 Ethics statement. The study protocol was approved by the Institutional Review Board of New
103 York University School of Medicine, Bellevue Hospital Center, and New York City Health and
104 Hospital Corporation for Activities Involving Human Subjects. Written informed consent was
105 obtained from all participants.
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107 Study participants. Twenty subjects were randomly selected from an HIV cohort study (16). lo
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108 Ten subjects who were seropositive for HIV and HAART naive or off therapy for at least 6 d
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109 months were recruited prior to the initiation of HAART, which was provided by the New York m
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110 University Clinical Trial Unit, Bellevue Hospital Center, New York. The other 10 subjects were t
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111 seronegative for HIV and were recruited from the Bluestone Center for Clinical Research, New jc
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112 York University College of Dentistry. Demographic data (for all subjects) and medical data (for a
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113 HIV+ subjects) were obtained from the medical records, including age, sex, ethnicity, HIV viral .o
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114 load, CD4+ and CD8+ T cell lymphocyte counts, and type and date of initiation of antiretroviral o/
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115 medications were collected and evaluated before and 6 months after therapy with HAART. For
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116 HIV- subjects, the status was confirmed using the OraQuick ADVANCE® Rapid HIV-1/2 il 3
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117 Antibody Test. 0
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118 b
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119 Oral examination and samples. At the initial evaluation, each subject received a u
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120 comprehensive oral examination by one of two standardized clinical examiners. Caries status
121 was determined at the tooth surface level according to criteria modified from the National Health
122 and Nutrition Examination Survey III, as well as an index of decayed and filled teeth (DFT) and
123 decayed and filled tooth surfaces (DFS) (28). Periodontal examination was performed for the 6
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124 Ramfjord index teeth (29) and at 6 sites on each tooth (mesiobuccal, buccal, distobuccal,
125 distolingual, lingual, and mesiolingual). Periodontal bleeding on probing (BOP) was recorded as
126 a dichotomous outcome for each site and deemed positive if bleeding occurred within 15 seconds
127 after the assessment of probing depth.
128 Stimulated whole saliva samples were collected for this study. In order to minimize
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129 potential variations, saliva sample collection was conducted in the morning, if possible. Each
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130 subject was asked to refrain from eating or drinking for at least 2 h prior to sample collection. lo
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131 After resting for 5 min with no talking, subjects were asked to rinse their mouth with sterile d
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132 water, then chew on a piece of paraffin wax for 30 seconds and expectorate directly into a m
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133 gradated 50 mL sample collection tube on ice. A portion of the saliva samples (2 mL) was t
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134 immediately transferred on ice to a microbiology laboratory (New York University College of jc
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135 Dentistry) and processed within 2 hours. For the HIV-seropositive subjects, a second saliva a
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136 sample was collected 6 months after starting treatment with HAART. .o
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137 o
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138 Microbial evaluation. Quantitative evaluation of oral microbial colonization and microbial
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139 diversity was performed with 3 methods: conventional culture, denaturing gradient gel il 3
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140 electrophoresis (DGGE), and human oral microbe identification microarray (HOMIM). 0
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141 Culture. Saliva samples were sonicated (30 seconds) and diluted (10-1 to 10-4). The b
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142 diluted samples (50 μL) were plated on selective media for cultivation of Streptococcus mutans u
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143 (mitis salivarius agar with potassium tellurite-bacitracin; Difco Laboratories Inc., Detroit, MI)
144 (30), Lactobacillus species (Rogosa, Thermo Scientific, Lenexa, KS) (31), total oral streptococci
145 (mitis salivarius agar; Anaerobe Systems, Morgan Hill, CA), and Candida species
146 (CHROMagar™ Candida; CHROMagar, Paris, France) (32, 33). The saliva samples were also
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147 plated on an enriched nonselective tryptic soy agar (ETSA; Anaerobe Systems, Morgan Hill, CA)
148 for cultivation of total cultivable oral bacteria. All bacterial samples were plated using Autoplate
149 4000 (Advanced Instruments, Norwood, MA) and incubated anaerobically (85% nitrogen, 10%
150 carbon dioxide, and 5% hydrogen) or aerobically for 72 hours, and colonies on each culture plate
151 were counted manually. For statistical analysis, the colony forming units (CFUs) were
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152 transformed logarithmically to normalize the variation in distribution and estimate the
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153 concentration of each targeted microorganism in saliva. The microbial CFUs were analyzed for lo
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154 association with viral load, CD4+ and CD8+ T cell counts, caries status (DFT/DFS score), and ed
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155 periodontal health status (BOP score). m
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156 Extraction of bacterial genomic DNA. Whole saliva (1 mL) was used for bacterial t
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157 genomic DNA extraction as previously described (34, 35). Briefly, the saliva sample was jc
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158 centrifuged (18 000 x g for 3 min). The supernatant was discarded, and total bacterial genomic a
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159 DNA was extracted from the pellet using a DNA purification kit (MasterPure, Epicentre, .o
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160 Madison, WI). Samples were treated with a solution of phenol, chloroform, and isoamyl alcohol o
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161 (25:24:1) at pH 8.0 and mutanolysin (5000 U/mL; 2 μL). DNA was precipitated from the A
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162 aqueous phase with isopropanol and recovered by centrifugation. The pellet was resuspended in 3
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163 20 μL of Tris-EDTA buffer. Final DNA quality and concentration was measured using the 1
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164 Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The bacterial DNA
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165 was stored at -20°C. ue
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166 16S rRNA gene amplification and denaturing gradient gel electrophoresis. This
167 assay is commonly used in studies of microbial ecology, microbiology, and environmental
168 microbiology (36-38). Previously, we have shown that the assay could provide a rapid
169 assessment of the oral bacterial community without cultivation, differentiate the major
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170 components of the microbial profile, and enable longitudinal monitoring of changes in microbial
171 flora in the same subjects (35, 39-42). In this study, we applied DGGE to all bacterial genomic
172 DNA samples. The complete 16S rRNA gene locus (1500 bp) was preamplified with universal
173 16S rRNA gene primers (8f to 1492r) as previously described (43). A second nested
174 amplification of the V4 to V5 hypervariable region of the 16S rRNA gene was performed using
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175 an internal set of primers (prbac1 and prbac2) (44) with a 40-nucleotide GC clamp added at the 5’
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176 -end of the forward primer (39, 45). After PCR amplification (GeneAmps PCR System 9700, PE lo
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177 Applied Biosystems, Foster City, CA), the PCR products (> 300 bp) were separated on linear d
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178 denaturing gradient polyacrylamide gels (40% to 60%) (DCode System, Bio-Rad, Hercules, CA). m
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179 Electrophoresis was performed (constant 60 V at 58°C for 16 h) in TAE buffer (Tris-acetate- t
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180 EDTA, pH 8.5). After electrophoresis, the gels were stained in ethidium bromide (0.5 mg/mL). jc
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181 The DGGE profile images were captured digitally using the AlphaImagerTM 3300 System a
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182 (Alpha Innotech, San Leandro, CA). .o
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183 Human oral microbe identification microarray assay (HOMIM). The bacterial o
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184 genomic DNA samples were analyzed for identification of specific microbial species using the
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185 human oral microbial identification microarray (HOMIM) (Forsyth Institute, Cambridge, MA). il 3
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186 The HOMIM assay allowed simultaneous detection of > 300 predominant oral bacterial species 0
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187 in a single hybridization experiment. The quality of the bacterial DNA was verified with PCR b
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188 using a new set of universal 16S rRNA primers (forward:AACTGGAGG-AAGGTGGGGAT; u
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189 reverse:AGGAGGTGATCCA-ACCGCA). A nested PCR reaction was performed to incorporate
190 the fluorescent materials (Cy3-dCTP) into the targeted DNA. The labeled 16S rRNA amplicons
191 were loaded on aldehyde-coated glass slides containing > 430 unique 16S rRNA-based reverse-
192 capture oligonucleotide probes that targeted >300 bacterial taxa (Forsyth Institute, Cambridge,
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193 MA) and were hybridized overnight at 55°C (46). The slides were washed with buffer (2x saline
194 sodium citrate, 10% sodium dodecyl sulfate) at 55°C, dried, and scanned (Axon GenePix 4000B
195 Microarray Scanner, Molecular Devices, Sunnyvale, CA). The raw data were normalized by
196 comparing individual signal intensities to the average signal from universal probes for 16S rRNA
197 genes (GenePix Pro software, Molecular Devices, LLC. Sunnyvale, CA) (46). The final
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198 microarray data were graded (range, 0 to 5) based on the presence or absence of the w
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199 hybridization signals and degree of signal intensity and imported to software for analysis (MeV a
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200 v4.8.1 software, Dana-Farber Cancer Institute, Boston, MA) (47). d
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201 m
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202 Statistical analysis. Bacterial CFUs for each targeted microorganism, viral load, CD4+ and tt
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203 CD8+ T cell counts, dental caries, and periodontal status scores were evaluated with jc
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204 nonparametric Mann-Whitney test for mean comparisons and Spearman rank correlation for s
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205 correlation analysis between HIV+ and HIV- groups, as well as within the HIV+ group before and .o
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206 after HAART. o
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207 The DGGE microbial flora profiles were analyzed with the BioNumerics 6.0 software p
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208 (Applied Maths, Austin, TX). Levels of similarity between the fingerprints were calculated 3
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209 based on the Dice coefficient of pairwise comparisons. Cluster analysis and dendrograms were 1
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210 constructed based on the Ward method and algorithm (48). Differences in the number of detected y
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211 DGGE bands and their frequency distribution patterns were determined and compared between e
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212 groups. Shannon diversity index was calculated on the relative abundance and evenness of the
213 16S gene fragments detected by DGGE.
214 HOMIM data were correlated to identify the relative abundance of oral microbial genes
215 of interest and compared among the three groups based on the presence or absence of genes and
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216 HOMIM hybridization intensity. Hierarchical clustering analysis was performed based on the
217 presence and absence of bands and the average linkage method (49). Dendrograms were
218 constructed by clustering correlation matrices that included all elements of gene comparisons.
219 An experimentwise false discovery rate (FDR) of 5% was used. The final P-values for the
220 analysis were adjusted for multiple comparisons using Holm's method (50). Principal
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221 Components Analysis was performed using the MultiExperiment Viewer (MeV v4.8.1) computer
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222 program. lo
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223 All data analyses were performed with statistical software (SAS/STAT software, SAS d
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224 Institute, Cary, NC; SPSS Statistics software, IBM Corp., Somers, NY). The nonparametric m
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225 Wilcoxon-Mann-Whitney test was used to compare the differences between HIV+ subjects and t
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226 HIV- controls. To compare HIV+ samples before and after HAART, Friedman's chi-square rank /jc
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227 test, equivalent to the Cochran-Mantel-Haenszel chi-square, was calculated. a
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228 .o
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229 RESULTS o
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230 HIV infection status. The schematic diagram of the study design was illustrated in Figure 1. No
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231 significant difference was observed in the mean age between the two groups. HIV-seropositive il 3
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232 subjects had more dental caries (mean DFS score), but similar levels of gingivitis and 0
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233 periodontitis (mean BOP score) (Table 1). Among the HIV+ subjects, HIV viral load ranged b
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234 from 9.0 x 103 to 7.1 x 105 copies/mL, but these results were markedly reduced for each subject u
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235 after therapy with HAART (P < 0.001; Mann-Whitney test) (Fig. 2A). At 6 months after therapy
236 with HAART, the mean CD4+ T cell count was increased by 36% (before therapy, 313 ± 192
237 cells/μL; after therapy, 426 ± 194 cells/μL), and the mean ratio of CD4+-to-CD8+ T cells
238 (CD4:CD8 ratio) was increased by 33% (before therapy, 43% ± 41%; after therapy, 57% ± 29%)
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