Subjects were patients of Dental Clinics at the University of Illinois College of Dentistry. All subjects provided written informed consent to participate in accordance with guidelines of the Office for the Protection of Research Subjects of the University of Illinois at Chicago, with formal approval of the study protocol, 2012-1030, by the Institutional Review Board 1 of the University of Illinois Chicago. This study was done in full accordance with the principles of the Declaration of Helsinki. Samles were taken only from sites that appeared normal in appearance in the clinic.
Mucosal sample collection and selection
For the PAH selection procedure swab samples were collected from the lateral border of the tongue, buccal, attached gingiva and oral pharynx. These were combined and placed in 3 mL Bushnell-Haas Broth (BHB) without glycerol. One milliliter culture was used to inoculate 9 mL BHB with 10 µg/mL each of benzo[a]pyrene, chrysene, fluoranthrene, naphthalene, phenanthrene, and pyrene. The culture was incubated with aeration for 3 weeks at 37°C. One hundred microliters were collected and plated on BHI agar plate and incubated for 48 h at 37°C in aerobic conditions. Samples used directly for 16S rRNA analysis were from gingiva or tongue and were immediately frozen in TE.
PAH degradation by microbes in liquid culture
C. albicans was grown overnight in YPD broth at 30°C with aeration. The positive control, Mycobacterium rutilum was prepared in BHI medium and incubated overnight at 30°C with aeration15. The overnight cultures of C. albicans and Mycobacterium rutilum were centrifuged, washed 3 times with BHB medium, and resuspended in BHB. Each culture was divided in 9 large glass test tubes, each containing 10 mL culture in BHB medium. Pyrene or phenanthrene was added (triplicates) to a final concentration of 20 µg/mL. BHB medium with microbial cells without PAH (triplicates) and BHB medium only served as controls. Tubes were incubated with aeration for 14 days in the dark at 30°C.
Identification of pyrene and phenanthrene metabolites
After the PAHs were incubated with the microbes, they were stored unopened at -80°C until analyzed by GC/MS. Once the samples thawed, the cultures were spiked with 20 µg of phenanthrene d-10 or pyrene d-10 prior to extraction to allow estimation of the recovery of the unreacted pyrene and phenanthrene. After spiking with the deuterated standard, the samples were vortexed and stored overnight in a refrigerator. Each of the conical glass vials containing samples were acidified to a pH of 2 and extracted three times with 3 mL of MTBE (methyl-tert butyl ether, a total of 9 mL). The MTBE/aqueous mixtures were centrifuged at 900 g for 10 minutes to separate the aqueous and MTBE layers. The MTBE extracts were combined and allowed to evaporate overnight in a standard hood and the following morning the remaining MTBE was evaporated under nitrogen. The test tubes containing the residual solid were rinsed (vortexed) with two, 400 µl aliquots of MTBE. The MTBE was transferred to an autosampler vial (with a borosilicate glass insert) and evaporated to dryness in a speed vac. This concentration and transfer step was repeated twice. Analytical procedures were designed to analyze oxidized (hydroxylated) phenanthrene and pyrene metabolites.
The samples were then derivatized (silylated) with N,O-Bis(trimethylsilyl)- trifluoroacetamide (BSTFA) with 1% trimethylsilyl chloride (TMSC) to convert the hydroxyl groups to trimethyl silyl groups. Extracts in the vials mentioned above were [10 µl BSTFA/TMCS in 200 µl CHCl3]. These samples were diluted 1/20 and 1/50 in chloroform and analyzed by GC/MS for the purpose of quantifying the unreacted PAHs. The most concentrated, undiluted samples were run as well for the purpose of identifying metabolites.
Derivatized extracts were analyzed using an Agilent 5977 Mass selective detector interfaced to an Agilent 7800 GC. A 30-meter Agilent DB5-MS column was used for the separation. The mobile phase was helium at a flow rate of 1 mL/min. The injector temperature was 240°C. A 20-min GC gradient was used for the separation with a helium flow rate of 1 mL/min. The initial temperature was held at 60°C for two minutes and then increased to 320°C at a rate of 20°/minute and held at 320°C for 10 min. Full scans were acquired for all analyses.
Genomic DNA was extracted from swabs using the MasterPure Gram Positive DNA Purification Kit (Epicentre, Madison, WI, USA) according to manufacturer’s instructions. For samples harvested from in vitro incubations sample DNA was extracted using alkaline lysis and Express Matrix purification (MP Biomedical, Santa Ana, CA, USA). Identification of clones was done after PCR amplification using consensus 16S rRNA gene specific primers, F1 and R1 or in the case of C. albicans 28S rRNA fungal specific primers NL-1 and NL-4.
Characterization of microbial community structure
Microbial community structure was characterized using high-throughput sequencing of PCR amplicons generated from the V1-V3 variable regions of bacterial 16S ribosomal RNA (rRNA) genes. Briefly, the widely used primer sets 27F/534R, targeting the V1-V3 variable region of the 16S rRNA gene of bacteria, were used for amplification as done earlier with slight modifications53.
A two-stage PCR or “targeted amplicon sequencing” (TAS) approach was performed to generate amplicon libraries, as described previously54,55. In the first of the TAS procedure, the templates were amplified (28 cycles) using primers containing 27F and 534R 16S rRNA gene sequence containing 5’ linkers CS1 and CS2 linkers, as described previously35. PCR were performed in 10 µl reaction volumes using the KAPA HiFi HotStart PCR Kit. The PCR conditions were as follows: 5 min initial denaturation at 95°C, followed by 28 cycles of: 95°C for 30”, 50°C for 30”, 72°C for 60”. Subsequently, a second PCR reaction was established, with 1 µl of amplification product from the first stage used as input to the second reaction.
The primers for the second stage amplifications were the AccessArray barcoding system primers (Fluidigm, San Francisco, CA, USA), containing Illumina sequencing adapters, sample-specific barcodes, and CS1 and CS2 linkers. PCR conditions for the second reaction were as follows: 5 min initial denaturation at 95°C, followed by 8 cycles of: 95°C for 30”, 60°C for 30”, 72°C for 60”. Samples were pooled in equimolar ratio and quantified using a Qubit 2.0 fluorometer.
Sequencing was performed on an Illumina MiSeq sequencer using standard V3 chemistry with paired-end, 300 base reads. Fluidigm sequencing primers, targeting the CS1 and CS2 linker regions, were used to initiate sequencing. Demultiplexing of reads was performed on instrument. Library preparation was performed at the DNA Services Facility at the University of Illinois at Chicago.
Raw paired-end FASTQ files were merged using the Paired-End reAd merger (PEAR) algorithm56. Merged data were then quality trimmed (Q20), and sequences shorter than 450 bases were removed. The remaining sequences were exported as FASTA and processed through the software package QIIME (v1.8.0)57. Sequences were screened for chimeras using the USEARCH61 algorithm and putative chimeric sequences were removed from the data set58. Chimera-free samples were then pooled, and clustered into operational taxonomic units (OTU) at 97% similarity using USEARCH assigned to taxonomic levels from phylum to species.
BIOMs were used to identify taxa which were significantly differentially abundant between a priori defined groups. Differences in microbiota taxonomic abundance between the groups were tested using Welch’s t-test using the software package STAMP59. Significance was set at P < 0.05. The Student t-test was used to compare number of distinct taxa isolated from smokers and nonsmokers, which takes into account that several smokers produced multiple taxa in the assay