Bioinformatic analysis of bacteria and host cell dual RNA-sequencing experiments.

Bacterial pathogens subvert host cells by manipulating cellular pathways for survival and replication; in turn, host cells respond to the invading pathogen through cascading changes in gene expression. Deciphering these complex temporal and spatial dynamics to identify novel bacterial virulence factors or host response pathways is crucial for improved diagnostics and therapeutics. Dual RNA sequencing (dRNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. This approach builds on the high sensitivity and resolution of RNA sequencing technology and is applicable to any bacteria that interact with eukaryotic cells, encompassing parasitic, commensal or mutualistic lifestyles. Several laboratory protocols have been presented that outline the collection, extraction and sequencing of total RNA for dRNA-Seq experiments, but there is relatively little guidance available for the detailed bioinformatic analyses required. This protocol outlines a typical dRNA-Seq experiment, based on a Chlamydia trachomatis-infected host cell, with a detailed description of the necessary bioinformatic analyses with currently available software tools.

Impact of Standard Bacterial Vaginosis Treatment on the Genital Microbiota, Immune Milieu, and Ex Vivo Human Immunodeficiency Virus Susceptibility.

BACKGROUND: Genital immunology is a key determinant of human immunodeficiency virus (HIV) susceptibility. Both factors are modulated by bacterial vaginosis (BV) and, to some extent, by Lactobacillus iners, the genital Lactobacillus spp. that predominates in African, Caribbean, and other Black (ACB) women. We conducted a clinical trial to assess the impact of oral metronidazole treatment on the genital immune parameters of HIV acquisition risks in Kenyan women with BV. METHODS: The primary endpoint was ex vivo cervical CD4+ T-cell HIV susceptibility after 1 month; secondary endpoints included genital cytokine/chemokine levels, cervical immune cell populations, and the composition of the cervico-vaginal microbiota by 16S ribosomal RNA gene amplicon sequencing. RESULTS: BV resolved (Nugent score ≤ 3) at 1 month in 20/45 participants, and cervical CD4+ T-cell HIV entry was moderately reduced in all participants, regardless of treatment outcome. Resolution of BV and reduced abundances of BV-associated gram-negative taxa correlated with reduced genital interleukin (IL)-1α/ß. However, BV resolution and the concomitant colonization by Lactobacillus iners substantially increased several genital chemokines associated with HIV acquisition, including interferon-γ inducible protein (IP)-10, macrophage inflammatory protein (MIP)-3α, and monokine induced by gamma interferon (MIG). In an independent cohort of ACB women, most of whom were BV-free, vaginal chemokines were again closely linked with L. iners abundance, though not other Lactobacillus spp. CONCLUSIONS: BV treatment reduced genital CD4+ T-cell HIV susceptibility and IL-1 levels, but dramatically increased the genital chemokines that may enhance HIV susceptibility; the latter effect was related to the restoration of an Lactobacillus iners-dominated microbiota. Further studies are needed before treatment of asymptomatic BV can be recommended for HIV prevention in ACB communities.

Group B Streptococcus and the Vaginal Microbiota.

Background: Streptococcus agalactiae (group B Streptococcus [GBS]) is an important neonatal pathogen and emerging cause of disease in adults. The major risk factor for neonatal disease is maternal vaginal colonization. However, little is known about the relationship between GBS and vaginal microbiota. Methods: Vaginal lavage samples from nonpregnant women were tested for GBS, and amplicon-based sequencing targeting the 16S ribosomal RNA V3-V4 region was performed. Results: Four hundred twenty-eight of 432 samples met the high-quality read threshold. There was no relationship between GBS carriage and demographic characteristics, α-diversity, or overall vaginal microbiota community state type (CST). Within the non-Lactobacillus-dominant CST IV, GBS positive status was significantly more prevalent in CST IV-A than CST IV-B. Significant clustering by GBS status was noted on principal coordinates analysis, and 18 individual taxa were found to be significantly associated with GBS carriage by linear discriminant analysis. After adjusting for race/ethnicity, 4 taxa were positively associated with GBS, and 6 were negatively associated. Conclusions: Vaginal microbiota CST and α-diversity are not related to GBS status. However, specific microbial taxa are associated with colonization of this important human pathogen, highlighting a potential role for the microbiota in promotion or inhibition of GBS colonization.

Phylogenetic analysis of human Chlamydia pneumoniae strains reveals a distinct Australian indigenous clade that predates European exploration of the continent.

BACKGROUND: The obligate intracellular bacterium Chlamydia pneumoniae is a common respiratory pathogen, which has been found in a range of hosts including humans, marsupials and amphibians. Whole genome comparisons of human C. pneumoniae have previously highlighted a highly conserved nucleotide sequence, with minor but key polymorphisms and additional coding capacity when human and animal strains are compared. RESULTS: In this study, we sequenced three Australian human C. pneumoniae strains, two of which were isolated from patients in remote indigenous communities, and compared them to all available C. pneumoniae genomes. Our study demonstrated a phylogenetically distinct human C. pneumoniae clade containing the two indigenous Australian strains, with estimates that the most recent common ancestor of these strains predates the arrival of European settlers to Australia. We describe several polymorphisms characteristic to these strains, some of which are similar in sequence to animal C. pneumoniae strains, as well as evidence to suggest that several recombination events have shaped these distinct strains. CONCLUSIONS: Our study reveals a greater sequence diversity amongst both human and animal C. pneumoniae strains, and suggests that a wider range of strains may be circulating in the human population than current sampling indicates.

Multidrug-resistant typhoid fever with neurologic findings on the Malawi-Mozambique border.

BACKGROUND: Salmonella enterica serovar Typhi causes an estimated 22 million cases of typhoid fever and 216 000 deaths annually worldwide. We investigated an outbreak of unexplained febrile illnesses with neurologic findings, determined to be typhoid fever, along the Malawi-Mozambique border. METHODS: The investigation included active surveillance, interviews, examinations of ill and convalescent persons, medical chart reviews, and laboratory testing. Classification as a suspected case required fever and ≥1 other finding (eg, headache or abdominal pain); a probable case required fever and a positive rapid immunoglobulin M antibody test for typhoid (TUBEX TF); a confirmed case required isolation of Salmonella Typhi from blood or stool. Isolates underwent antimicrobial susceptibility testing and subtyping by pulsed-field gel electrophoresis (PFGE). RESULTS: We identified 303 cases from 18 villages with onset during March-November 2009; 214 were suspected, 43 were probable, and 46 were confirmed cases. Forty patients presented with focal neurologic abnormalities, including a constellation of upper motor neuron signs (n = 19), ataxia (n = 22), and parkinsonism (n = 8). Eleven patients died. All 42 isolates tested were resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole; 4 were also resistant to nalidixic acid. Thirty-five of 42 isolates were indistinguishable by PFGE. CONCLUSIONS: The unusual neurologic manifestations posed a diagnostic challenge that was resolved through rapid typhoid antibody testing in the field and subsequent blood culture confirmation in the Malawi national reference laboratory. Extending laboratory diagnostic capacity, including blood culture, to populations at risk for typhoid fever in Africa will improve outbreak detection, response, and clinical treatment.

Insight into the ecology of vaginal bacteria through integrative analyses of metagenomic and metatranscriptomic data.

BACKGROUND: Vaginal bacterial communities dominated by Lactobacillus species are associated with a reduced risk of various adverse health outcomes. However, somewhat unexpectedly, many healthy women have microbiota that are not dominated by lactobacilli. To determine the factors that drive vaginal community composition we characterized the genetic composition and transcriptional activities of vaginal microbiota in healthy women. RESULTS: We demonstrate that the abundance of a species is not always indicative of its transcriptional activity and that impending changes in community composition can be predicted from metatranscriptomic data. Functional comparisons highlight differences in the metabolic activities of these communities, notably in their degradation of host produced mucin but not glycogen. Degradation of mucin by communities not dominated by Lactobacillus may play a role in their association with adverse health outcomes. Finally, we show that the transcriptional activities of L. crispatus, L. iners, and Gardnerella vaginalis vary with the taxonomic composition of the communities in which they reside. Notably, L. iners and G. vaginalis both demonstrate lower expression of their cholesterol-dependent cytolysins when co-resident with Lactobacillus spp. and higher expression when co-resident with other facultative and obligate anaerobes. The pathogenic potential of these species may depend on the communities in which they reside and thus could be modulated by interventional strategies. CONCLUSIONS: Our results provide insight to the functional ecology of the vaginal microbiota, demonstrate the diagnostic potential of metatranscriptomic data, and reveal strategies for the management of these ecosystems.

Characterization of toxigenic Vibrio cholerae from Haiti, 2010-2011.

In October 2010, the US Centers for Disease Control and Prevention received reports of cases of severe watery diarrhea in Haiti. The cause was confirmed to be toxigenic Vibrio cholerae, serogroup O1, serotype Ogawa, biotype El Tor. We characterized 122 isolates from Haiti and compared them with isolates from other countries. Antimicrobial drug susceptibility was tested by disk diffusion and broth microdilution. Analyses included identification of rstR and VC2346 genes, sequencing of ctxAB and tcpA genes, and pulsed-field gel electrophoresis with SfiI and NotI enzymes. All isolates were susceptible to doxycycline and azithromycin. One pulsed-field gel electrophoresis pattern predominated, and ctxB sequence of all isolates matched the B-7 allele. We identified the tcpETCIRS allele, which is also present in Bangladesh strain CIRS 101. These data show that the isolates from Haiti are clonally and genetically similar to isolates originating in Africa and southern Asia and that ctxB-7 and tcpET(CIRS) alleles are undergoing global dissemination.

Dual RNA-seq analysis of in vitro infection multiplicity and RNA depletion methods in Chlamydia-infected epithelial cells.

Dual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods. Here, we have applied dual RNA-seq to Chlamydia trachomatis infected epithelial cells to examine transcriptomic responses from both organisms. We compared two time points post infection (1 and 24 h), three multiplicity of infection (MOI) ratios (0.1, 1 and 10) and two RNA depletion methods (rRNA and polyA). Capture of bacterial-specific RNA were greatest when combining rRNA and polyA depletion, and when using a higher MOI. However, under these conditions, host RNA capture was negatively impacted. Although it is tempting to use high infection rates, the implications on host cell survival, the potential reduced length of infection cycles and real world applicability should be considered. This data highlights the delicate nature of balancing host-pathogen RNA capture and will assist future transcriptomic-based studies to achieve more specific and relevant infection-related biological insights.

NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility.

The Escherichia coli NsrR protein is a nitric oxide-sensitive repressor of transcription. The NsrR-binding site is predicted to comprise two copies of an 11 bp motif arranged as an inverted repeat with 1 bp spacing. By mutagenesis we confirmed that both 11 bp motifs are required for maximal NsrR repression of the ytfE promoter. We used chromatin immunoprecipitation and microarray analysis (ChIP-chip) to show that NsrR binds to 62 sites close to the 5' ends of genes. Analysis of the ChIP-chip data suggested that a single 11 bp motif (with the consensus sequence AANATGCATTT) can function as an NsrR-binding site in vivo. NsrR binds to sites in the promoter regions of the fliAZY, fliLMNOPQR and mqsR-ygiT transcription units, which encode proteins involved in motility and biofilm development. Reporter fusion assays confirmed that NsrR negatively regulates the fliA and fliL promoters. A mutation in the predicted 11 bp NsrR-binding site in the fliA promoter impaired repression by NsrR and prevented detectable binding in vivo. Assays on soft-agar confirmed that NsrR is a negative regulator of motility in E. coli K12 and in a uropathogenic strain; surface attachment assays revealed decreased levels of attached growth in the absence of NsrR.

Chromatin accessibility dynamics of Chlamydia-infected epithelial cells.

Chlamydia are Gram-negative, obligate intracellular bacterial pathogens responsible for a broad spectrum of human and animal diseases. In humans, Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and is the causative agent of trachoma (infectious blindness) in disadvantaged populations. Over the course of its developmental cycle, Chlamydia extensively remodels its intracellular niche and parasitises the host cell for nutrients, with substantial resulting changes to the host cell transcriptome and proteome. However, little information is available on the impact of chlamydial infection on the host cell epigenome and global gene regulation. Regions of open eukaryotic chromatin correspond to nucleosome-depleted regions, which in turn are associated with regulatory functions and transcription factor binding. We applied formaldehyde-assisted isolation of regulatory elements enrichment followed by sequencing (FAIRE-Seq) to generate temporal chromatin maps of C. trachomatis-infected human epithelial cells in vitro over the chlamydial developmental cycle. We detected both conserved and distinct temporal changes to genome-wide chromatin accessibility associated with C. trachomatis infection. The observed differentially accessible chromatin regions include temporally-enriched sets of transcription factors, which may help shape the host cell response to infection. These regions and motifs were linked to genomic features and genes associated with immune responses, re-direction of host cell nutrients, intracellular signalling, cell-cell adhesion, extracellular matrix, metabolism and apoptosis. This work provides another perspective to the complex response to chlamydial infection, and will inform further studies of transcriptional regulation and the epigenome in Chlamydia-infected human cells and tissues.

A comprehensive non-redundant gene catalog reveals extensive within-community intraspecies diversity in the human vagina.

Analysis of metagenomic and metatranscriptomic data is complicated and typically requires extensive computational resources. Leveraging a curated reference database of genes encoded by members of the target microbiome can make these analyses more tractable. In this study, we assemble a comprehensive human vaginal non-redundant gene catalog (VIRGO) that includes 0.95 million non-redundant genes. The gene catalog is functionally and taxonomically annotated. We also construct a vaginal orthologous groups (VOG) from VIRGO. The gene-centric design of VIRGO and VOG provides an easily accessible tool to comprehensively characterize the structure and function of vaginal metagenome and metatranscriptome datasets. To highlight the utility of VIRGO, we analyze 1,507 additional vaginal metagenomes, and identify a high degree of intraspecies diversity within and across vaginal microbiota. VIRGO offers a convenient reference database and toolkit that will facilitate a more in-depth understanding of the role of vaginal microorganisms in women's health and reproductive outcomes.

VALENCIA: a nearest centroid classification method for vaginal microbial communities based on composition.

BACKGROUND: Taxonomic profiles of vaginal microbial communities can be sorted into a discrete number of categories termed community state types (CSTs). This approach is advantageous because collapsing a hyper-dimensional taxonomic profile into a single categorical variable enables efforts such as data exploration, epidemiological studies, and statistical modeling. Vaginal communities are typically assigned to CSTs based on the results of hierarchical clustering of the pairwise distances between samples. However, this approach is problematic because it complicates between-study comparisons and because the results are entirely dependent on the particular set of samples that were analyzed. We sought to standardize and advance the assignment of samples to CSTs. RESULTS: We developed VALENCIA (VAginaL community state typE Nearest CentroId clAssifier), a nearest centroid-based tool which classifies samples based on their similarity to a set of reference centroids. The references were defined using a comprehensive set of 13,160 taxonomic profiles from 1975 women in the USA. This large dataset allowed us to comprehensively identify, define, and characterize vaginal CSTs common to reproductive age women and expand upon the CSTs that had been defined in previous studies. We validated the broad applicability of VALENCIA for the classification of vaginal microbial communities by using it to classify three test datasets which included reproductive age eastern and southern African women, adolescent girls, and a racially/ethnically and geographically diverse sample of postmenopausal women. VALENCIA performed well on all three datasets despite the substantial variations in sequencing strategies and bioinformatics pipelines, indicating its broad application to vaginal microbiota. We further describe the relationships between community characteristics (vaginal pH, Nugent score) and participant demographics (race, age) and the CSTs defined by VALENCIA. CONCLUSION: VALENCIA provides a much-needed solution for the robust and reproducible assignment of vaginal community state types. This will allow unbiased analysis of both small and large vaginal microbiota datasets, comparisons between datasets and meta-analyses that combine multiple datasets. Video abstract.

Dual RNA-Seq of Chlamydia and Host Cells.

During the infection of a host cell by a bacterial pathogen, a cascading series of gene expression changes occurs as each organism manipulates or responds to the other via defense or survival strategies. Unraveling this complex interplay is key for our understanding of bacterial virulence and host response pathways for the development of novel therapeutics. Dual RNA sequencing (dual RNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. Leveraging the sensitivity and resolution allowed by RNA-seq, dual RNA-Seq can be applied to any bacteria-eukaryotic host interaction. We pioneered dual RNA-Seq to simultaneously capture Chlamydia and host expression profiles during an in vitro infection as proof of principle. Here we provide a detailed laboratory protocol and bioinformatics analysis guidelines for dual RNA-seq experiments focusing on Chlamydia as the organism of interest.

Cervicovaginal microbiota and local immune response modulate the risk of spontaneous preterm delivery.

Failure to predict and understand the causes of preterm birth, the leading cause of neonatal morbidity and mortality, have limited effective interventions and therapeutics. From a cohort of 2000 pregnant women, we performed a nested case control study on 107 well-phenotyped cases of spontaneous preterm birth (sPTB) and 432 women delivering at term. Using innovative Bayesian modeling of cervicovaginal microbiota, seven bacterial taxa were significantly associated with increased risk of sPTB, with a stronger effect in African American women. However, higher vaginal levels of ß-defensin-2 lowered the risk of sPTB associated with cervicovaginal microbiota in an ethnicity-dependent manner. Surprisingly, even in Lactobacillus spp. dominated cervicovaginal microbiota, low ß-defensin-2 was associated with increased risk of sPTB. These findings hold promise for diagnostics to accurately identify women at risk for sPTB early in pregnancy. Therapeutic strategies could include immune modulators and microbiome-based therapeutics to reduce this significant health burden.

Early Transcriptional Landscapes of Chlamydia trachomatis-Infected Epithelial Cells at Single Cell Resolution.

Chlamydia are Gram-negative obligate intracellular bacterial pathogens responsible for a variety of disease in humans and animals worldwide. Chlamydia trachomatis causes trachoma in disadvantaged populations, and is the most common bacterial sexually transmitted infection in humans, causing reproductive tract disease. Antibiotic therapy successfully treats diagnosed chlamydial infections, however asymptomatic infections are common. High-throughput transcriptomic approaches have explored chlamydial gene expression and infected host cell gene expression. However, these were performed on large cell populations, averaging gene expression profiles across all cells sampled and potentially obscuring biologically relevant subsets of cells. We generated a pilot dataset, applying single cell RNA-Seq (scRNA-Seq) to C. trachomatis infected and mock-infected epithelial cells to assess the utility, pitfalls and challenges of single cell approaches applied to chlamydial biology, and to potentially identify early host cell biomarkers of chlamydial infection. Two hundred sixty-four time-matched C. trachomatis-infected and mock-infected HEp-2 cells were collected and subjected to scRNA-Seq. After quality control, 200 cells were retained for analysis. Two distinct clusters distinguished 3-h cells from 6- and 12-h. Pseudotime analysis identified a possible infection-specific cellular trajectory for Chlamydia-infected cells, while differential expression analyses found temporal expression of metallothioneins and genes involved with cell cycle regulation, innate immune responses, cytoskeletal components, lipid biosynthesis and cellular stress. We find that changes to the host cell transcriptome at early times of C. trachomatis infection are readily discernible by scRNA-Seq, supporting the utility of single cell approaches to identify host cell biomarkers of chlamydial infection, and to further deconvolute the complex host response to infection.

Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform.

Amplification, sequencing, and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300-bp paired-end reads of higher quality than those produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-step PCR amplification protocol is also described that allows for targeting of different amplicon regions, and enhances amplification success from samples with low bacterial bioburden. IMPORTANCE Amplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high-throughput sequencing have made it a widely adopted approach, especially for projects that necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per-sample cost relative to those of the Illumina MiSeq platform without sacrificing amplicon length. To make this method more flexible for various amplicon-targeted regions as well as improve amplification from low-biomass samples, we also present and validate a 2-step PCR library preparation method.

The Cervicovaginal Microbiota-Host Interaction Modulates Chlamydia trachomatis Infection.

The mechanism(s) by which Lactobacillus-dominated cervicovaginal microbiota provide a barrier to Chlamydia trachomatis infection remain(s) unknown. Here we evaluate the impact of different Lactobacillus spp. identified via culture-independent metataxonomic analysis of C. trachomatis-infected women on C. trachomatis infection in a three-dimensional (3D) cervical epithelium model. Lactobacillus spp. that specifically produce d(-) lactic acid were associated with long-term protection against C. trachomatis infection, consistent with reduced protection associated with Lactobacillus iners, which does not produce this isoform, and with decreased epithelial cell proliferation, consistent with the observed prolonged protective effect. Transcriptomic analysis revealed that epigenetic modifications involving histone deacetylase-controlled pathways are integral to the cross talk between host and microbiota. These results highlight a fundamental mechanism whereby the cervicovaginal microbiota modulates host functions to protect against C. trachomatis infection.IMPORTANCE The vaginal microbiota is believed to protect women against Chlamydia trachomatis, the etiologic agent of the most prevalent sexually transmitted infection (STI) in developed countries. The mechanism underlying this protection has remained elusive. Here, we reveal the comprehensive strategy by which the cervicovaginal microbiota modulates host functions to protect against chlamydial infection, thereby providing a novel conceptual mechanistic understanding. Major implications of this work are that (i) the impact of the vaginal microbiota on the epithelium should be considered in future studies of chlamydial infection and other STIs and (ii) a fundamental understanding of the cervicovaginal microbiota's role in protection against STIs may enable the development of novel microbiome-based therapeutic strategies to protect women from infection and improve vaginal and cervical health.

A high-throughput sequencing assay to comprehensively detect and characterize unicellular eukaryotes and helminths from biological and environmental samples.

BACKGROUND: Several of the most devastating human diseases are caused by eukaryotic parasites transmitted by arthropod vectors or through food and water contamination. These pathogens only represent a fraction of all unicellular eukaryotes and helminths that are present in the environment and many uncharacterized organisms might have subtle but pervasive effects on health, including by modifying the microbiome where they reside. Unfortunately, while we have modern molecular tools to characterize bacterial and, to a lesser extent, fungal communities, we lack suitable methods to comprehensively investigate and characterize most unicellular eukaryotes and helminths: the detection of these organisms often relies on microscopy that cannot differentiate related organisms, while molecular assays can only detect the pathogens specifically tested. RESULTS: Here, we describe a novel sequencing-based assay, akin to bacterial 16S rRNA sequencing, that enables high-throughput detection and characterization of a wide range of unicellular eukaryotes and helminths, including those from taxonomical groups containing all common human parasites. We designed and evaluated taxon-specific PCR primer pairs that selectively amplify all species from eight taxonomical groups (Apicomplexa, Amoeba, Diplomonadida, Kinetoplastida, Parabasalia, Nematoda, Platyhelminthes, and Microsporidia). We then used these primers to screen DNA extracted from clinical, biological, and environmental samples, and after next-generation sequencing, identified both known and previously undescribed organisms from most taxa targeted. CONCLUSIONS: This novel high-throughput assay enables comprehensive detection and identification of eukaryotic parasites and related organisms, from a wide range of complex biological and environmental samples. This approach can be easily deployed to many settings and will efficiently complement existing methods and provide a holistic perspective on the microbiome.

A Laboratory Methodology for Dual RNA-Sequencing of Bacteria and their Host Cells In Vitro.

Dual RNA-Sequencing leverages established next-generation sequencing (NGS)-enabled RNA-Seq approaches to measure genome-wide transcriptional changes of both an infecting bacteria and host cells. By simultaneously investigating both organisms from the same biological sample, dual RNA-Seq can provide unique insight into bacterial infection processes and reciprocal host responses at once. However, the difficulties involved in handling both prokaryotic and eukaryotic material require distinct, optimized procedures. We previously developed and applied dual RNA-Seq to measure prokaryotic and eukaryotic expression profiles of human cells infected with bacteria, using in vitro Chlamydia-infected epithelial cells as proof of principle. Here we provide a detailed laboratory protocol for in vitro dual RNA-Seq that is readily adaptable to any host-bacteria system of interest.

The effect of cigarette smoking on the oral and nasal microbiota.

BACKGROUND: The goal of the study was to investigate whether cigarette smoking alters oral and nasal microbial diversity, composition, and structure. Twenty-three current smokers and 20 never smokers were recruited. From each subject, nine samples including supra and subgingiva plaque scrapes, saliva, swabs from five soft oral tissue sites, and one nasal swab from both the anterior nares were collected. 16S rRNA V3-V4 region was sequenced for microbial profiles. RESULTS: We found that alpha diversity was lower in smokers than in nonsmokers in the buccal mucosa, but in other sample sites, microbial diversity and composition were not significantly different by smoking status. Microbial profiles differed significantly among eight oral sites. CONCLUSIONS: This study investigates the effect of cigarette smoking on different sites of the oral cavity and shows a potential effect of cigarette smoking on the buccal mucosa microbiota. The marked heterogeneity of the oral microbial ecosystem that we found may contribute to the stability of the oral microbiota in most sites when facing environmental perturbations such as that caused by cigarette smoking.