HtrA, fatty acids, and membrane protein interplay in Chlamydia trachomatis to impact stress response and trigger early cellular exit.

Chlamydia trachomatis is an intracellular bacterial pathogen that undergoes a biphasic developmental cycle, consisting of intracellular reticulate bodies and extracellular infectious elementary bodies. A conserved bacterial protease, HtrA, was shown previously to be essential for Chlamydia during the reticulate body phase, using a novel inhibitor (JO146). In this study, isolates selected for the survival of JO146 treatment were found to have polymorphisms in the acyl-acyl carrier protein synthetase gene (aasC). AasC encodes the enzyme responsible for activating fatty acids from the host cell or synthesis to be incorporated into lipid bilayers. The isolates had distinct lipidomes with varied fatty acid compositions. A reduction in the lipid compositions that HtrA prefers to bind to was detected, yet HtrA and MOMP (a key outer membrane protein) were present at higher levels in the variants. Reduced progeny production and an earlier cellular exit were observed. Transcriptome analysis identified that multiple genes were downregulated in the variants especially stress and DNA processing factors. Here, we have shown that the fatty acid composition of chlamydial lipids, HtrA, and membrane proteins interplay and, when disrupted, impact chlamydial stress response that could trigger early cellular exit. IMPORTANCE: Chlamydia trachomatis is an important obligate intracellular pathogen that has a unique biphasic developmental cycle. HtrA is an essential stress or virulence protease in many bacteria, with many different functions. Previously, we demonstrated that HtrA is critical for Chlamydia using a novel inhibitor. In the present study, we characterized genetic variants of Chlamydia trachomatis with reduced susceptibility to the HtrA inhibitor. The variants were changed in membrane fatty acid composition, outer membrane proteins, and transcription of stress genes. Earlier and more synchronous cellular exit was observed. Combined, this links stress response to fatty acids, membrane proteins, and HtrA interplay with the outcome of disrupted timing of chlamydial cellular exit.

Phylogenomic analysis of a global collection of Escherichia coli ST38: evidence of interspecies and environmental transmission?

IMPORTANCE: Extraintestinal pathogenic Escherichia coli (ExPEC) sequence type (ST) 38 is one of the top 10 human pandemic lineages. Although a major cause of urinary tract and blood stream infections, ST38 has been poorly characterized from a global phylogenomic perspective. A comprehensive genome-scale analysis of 925 ST38 isolate genomes identified two broad ancestral clades and linkage of discrete ST38 clusters with specific bla CTX-M variants. In addition, the clades and clusters carry important virulence genes, with diverse but poorly characterized plasmids. Numerous putative interhost and environment transmission events were identified here by the presence of ST38 clones (defined as isolates with ≤35 SNPs) within humans, companion animals, food sources, urban birds, wildlife, and the environment. A small cluster of international ST38 clones from diverse sources, likely representing progenitors of a hospital outbreak that occurred in Brisbane, Australia, in 2017, was also identified. Our study emphasizes the importance of characterizing isolate genomes derived from nonhuman sources and geographical locations, without any selection bias.

Genome organization and genomics in Chlamydia: whole genome sequencing increases understanding of chlamydial virulence, evolution, and phylogeny.

The genus Chlamydia contains important obligate intracellular bacterial pathogens to humans and animals, including C. trachomatis and C. pneumoniae. Since 1998, when the first Chlamydia genome was published, our understanding of how these microbes interact, evolved and adapted to different intracellular host environments has been transformed due to the expansion of chlamydial genomes. This review explores the current state of knowledge in Chlamydia genomics and how whole genome sequencing has revolutionised our understanding of Chlamydia virulence, evolution, and phylogeny over the past two and a half decades. This review will also highlight developments in multi-omics and other approaches that have complemented whole genome sequencing to advance knowledge of Chlamydia pathogenesis and future directions for chlamydial genomics.

Phylogenomics of two ST1 antibiotic-susceptible non-clinical Acinetobacter baumannii strains reveals multiple lineages and complex evolutionary history in global clone 1.

Acinetobacter baumannii is an opportunistic pathogen that is difficult to treat due to its resistance to extreme conditions, including desiccation and antibiotics. Most strains causing outbreaks around the world belong to two main global lineages, namely global clones 1 and 2 (GC1 and GC2). Here, we used a combination of Illumina short read and MinION (Oxford Nanopore) long-read sequence data with a hybrid assembly approach to complete the genome sequence of two antibiotic-sensitive GC1 strains, Ex003 and Ax270, recovered in Lebanon from water and a rectal swab of a cat, respectively. Phylogenetic analysis of Ax270 and Ex003 with 186 publicly available GC1 genomes revealed two major clades, including five main lineages (L1-L5), and four single-isolate lineages outside of the two clades. Ax270 and Ex003, along with AB307-0294 and MRSN7213 (both predicted antibiotic-susceptible isolates) represent these individual lineages. Antibiotic resistance islands and transposons interrupting the comM gene remain important features in L1-L5, with L1 associated with the AbaR-type resistance islands, L2 with AbaR4, L3 strains containing either AbaR4 or its variants as well as Tn6022::ISAba42, and L4 and L5 associated with Tn6022 or its variants. Analysis of the capsule (KL) and outer core (OCL) polysaccharide loci further revealed a complex evolutionary history probably involving many recombination events. As more genomes become available, more GC1 lineages continue to emerge. However, genome sequence data from more diverse geographical regions are needed to draw a more accurate population structure of this globally distributed clone.

Two carbapenem-resistant ST1:ST231:KL1:OCL1 Acinetobacter baumannii strains recovered in Tehran, Iran, carry AbaR31 in the chromosome and AbaR4 and TnaphA6 in a RepAci6 plasmid.

OBJECTIVES: To analyse the context of genes conferring antibiotic resistance in two carbapenem-resistant Acinetobacter baumannii isolates recovered in Tehran, Iran. METHODS: The antibiotic resistance phenotype for 28 antibiotics was determined using disc diffusion. The whole genome sequences of ABH008 and ABS200 were determined using the Illumina HiSeq X Ten platform. Resistance genes were identified using ResFinder and multilocus sequence types were determined using the Oxford and Institut Pasteur schemes. RESULTS: Isolates ABH008 and ABS200, recovered in 2012 and 2013, respectively, in two different Tehran hospitals, belong to the common global clone 1 lineage, ST1IP and ST231OX. They are resistant to sulfamethoxazole, tetracycline, gentamicin, amikacin, third-generation cephalosporins and carbapenems. Despite being isolated in different hospitals, phylogenetic analysis indicated they are closely related. Consistent with this, both isolates carry catA1, sul1, aacC1 and aadA1 in a novel variant of the AbaR3-type resistance island, named AbaR31. Both isolates are resistant to amikacin and carbapenems owing to aphA6 and oxa23, respectively. The oxa23 gene is located in the AbaR4 resistance island, and aphA6 in TnaphA6, and both mobile elements are in an ∼90 kbp plasmid encoding the putative RepAci6 replication initiation protein. Resistance to third-generation cephalosporins is due to the acquisition by homologous recombination of a 5 kb DNA segment that contains ISAba1-ampC from a ST623 strain. CONCLUSIONS: The resistance gene complements of ABH008 and ABS200 were found in AbaR31 and a plasmid that encodes RepAci6. The close genetic relationship of ABH008 and ABS200, despite each being recovered from different hospitals, indicates transmission between the two hospitals.

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.

Antibiotics and antimicrobial resistance: Evaluation of the knowledge, attitude, and perception among students and faculty within US dental schools.

PURPOSE: This study examined knowledge, attitudes, perceptions, and awareness regarding antibiotic use among students and academic faculty in US dental schools. METHODS: Two questionnaires, 1 for third-year/fourth-year dental students and the other for academic deans/department chairs were administered electronically. Questions on demographics, antibiotic knowledge, educational formats, and the role of dentistry in antibiotic stewardship were included. Knowledge about antibiotics and antibiotics stewardship was compared between third-year and fourth-year students and between students and academic faculty using t-test and chi-squared test at 0.05 significance level. RESULTS: A total of 18 responses on the academic dean and department chair survey and 172 responses on the dental student survey were collected. Overall, 71% of students reported that they could benefit from more education regarding antibiotics. Both faculty and students agreed that dentistry should play an important role in reducing antimicrobial resistance, but most dental students were "not at all familiar" with the term antimicrobial stewardship and several (32%) were unsure if clinical guidelines were present at their schools. CONCLUSION: Improvements to the dental educational curriculum regarding the responsible use of antibiotics, along with the implementation of stewardship programs within dentistry are strongly encouraged.

Denosumab Use as a Predictor Variable for External Cervical Resorption: A Case-Control Study.

INTRODUCTION: The objective of this case-control study was to investigate the association between denosumab use and the risk of developing external cervical resorption (ECR). METHODS: Thirty-three patients ≥45 years old who were diagnosed with ECR were selected. Controls were matched to the cases based on sex and age (±5 years) in a 1:1 ratio. Confounders were classified into systemic factors, including a history of systemic sclerosis, hepatitis B, denosumab use, and bisphosphonate use, or local factors, including a history of traumatic occlusion, periodontal procedures (scaling and root planing and periodontal surgeries), and tooth extraction (excluding third molar extraction). Additionally, the number of remaining teeth in each subject was recorded using panoramic radiographs. The baseline characteristics of the 2 groups, including age, sex, and the number of remaining teeth, were compared using the chi-square and Mann-Whitney U tests. Binary logistic regression was used to determine the possible association between denosumab use and the risk of developing ECR (α < 0.05). RESULTS: No significant differences in baseline characteristics were observed between the case and control groups (P > .05). After adjusting for systemic and local cofounders, denosumab use was significantly associated with the occurrence of ECR (odds ratio = 7.317; 95% confidence interval, 1.410-37.966; P < .05). CONCLUSIONS: Based on the binary logistic regression model, denosumab use could significantly predict the risk of developing ECR.

Analysis of Complete Genome Sequence of Acinetobacter baumannii Strain ATCC 19606 Reveals Novel Mobile Genetic Elements and Novel Prophage.

Acinetobacter baumannii isolate ATCC 19606 was recovered in the US prior to 1948. It has been used as a reference and model organism in many studies involving antibiotic resistance and pathogenesis of A. baumannii, while, until recently, a complete genome of this strain was not available. Here, we present an analysis of the complete 3.91-Mbp genome sequence, generated via a combination of short-read sequencing (Illumina) and long-read sequencing (MinION), and show it contains two small cryptic plasmids and a novel complete prophage of size 41.2 kb. We also characterised several regions of the ATCC 19606 genome, leading to the identification of a novel cadmium/mercury transposon, which was named Tn6551. ATCC 19606 is an antibiotic-sensitive strain, but a comparative analysis of all publicly available ST52 strains predicts a resistance to modern antibiotics by the accumulation of antibiotic-resistance genes via plasmids in recent isolates that belong to this sequence type.

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.

Dynamically Navigated versus Freehand Access Cavity Preparation: A Comparative Study on Substance Loss Using Simulated Calcified Canals.

INTRODUCTION: The aim of this in vitro study was to compare the speed, qualitative precision, and quantitative loss of tooth structure with freehand and dynamically navigated access preparation techniques for root canal location in 3-dimensional-printed teeth with simulated calcified root canals. METHODS: Forty maxillary and mandibular central incisors (tooth #9 and tooth #25) were 3-dimensionally printed to simulate canal calcification. Under simulated clinical conditions, access preparations were randomly performed with contemporary freehand and dynamically navigated techniques. Qualitative precision and quantitative loss of tooth structure were assessed on postoperative cone-beam computed tomographic scans using ITK-SNAP open-source segmentation (http://www.itksnap.org/). The associations between jaw, technique, volume of substance loss, and operating time were determined using analysis of variance models with Tukey-adjusted post hoc pair-wise comparisons. The kappa statistic was used to determine agreement between 2 independent, blinded raters on the qualitative assessment of the drill path. The association between the technique and jaw and qualitative assessment scoring was compared using the Fisher exact test. The significance level was set at .05. RESULTS: Dynamically navigated accesses resulted in significantly less mean substance loss in comparison with the freehand technique (27.2 vs 40.7 mm3, P < .05). Dynamically navigated accesses were also associated with higher optimal precision (drill path centered) to locate calcified canals in comparison with the freehand technique (75% vs 45%, P > .05). Mandibular teeth were associated with a negligible difference in substance loss between the access techniques (19.0 vs 19.1 mm3, P > .05). However, qualitatively the freehand technique was still prone to 30% higher chance of suboptimal precision (drill path tangentially transported) in locating calcified canals. Overall, dynamically navigated accesses were prepared significantly faster than freehand preparations (2.2 vs 7.06 minutes, P < .05). CONCLUSIONS: Within the limitations of this in vitro study, overall dynamically navigated access preparations led to significantly less mean substance loss with optimal and efficient precision in locating simulated anterior calcified root canals in comparison with freehand access preparations.

Escherichia coli ST8196 is a novel, locally evolved, and extensively drug resistant pathogenic lineage within the ST131 clonal complex.

The H30Rx subclade of Escherichia coli ST131 is a clinically important, globally dispersed pathogenic lineage that typically displays resistance to fluoroquinolones and extended spectrum ß-lactams. Isolates EC233 and EC234, variants of ST131-H30Rx with a novel sequence type (ST) 8196, isolated from unrelated patients presenting with bacteraemia at a Sydney Hospital in 2014 are characterised here. EC233 and EC234 are phylogroup B2, serotype O25:H4A, and resistant to ampicillin, amoxicillin, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, norfloxacin and gentamicin and are likely clonal. Both harbour an IncFII_2 plasmid (pSPRC_Ec234-FII) that carries most of the resistance genes on an IS26 associated translocatable unit, two small plasmids and a novel IncI1 plasmid (pSPRC_Ec234-I). SNP-based phylogenetic analysis of the core genome of representatives within the ST131 clonal complex places both isolates in a subclade with three clinical Australian ST131-H30Rx clade-C isolates. A MrBayes phylogeny analysis of EC233 and EC234 indicates ST8196 share a most recent common ancestor with ST131-H30Rx strain EC70 isolated from the same hospital in 2013. Our study identified genomic hallmarks that define the ST131-H30Rx subclade in the ST8196 isolates and highlights a need for unbiased genomic surveillance approaches to identify novel high-risk MDR E. coli pathogens that impact healthcare facilities.

Genomic profiling of Escherichia coli isolates from bacteraemia patients: a 3-year cohort study of isolates collected at a Sydney teaching hospital.

This study sought to assess the genetic variability of Escherichia coli isolated from bloodstream infections (BSIs) presenting at Concord Hospital, Sydney during 2013-2016. Whole-genome sequencing was used to characterize 81 E. coli isolates sourced from community-onset (CO) and hospital-onset (HO) BSIs. The cohort comprised 64 CO and 17 HO isolates, including 35 multidrug-resistant (MDR) isolates exhibiting phenotypic resistance to three or more antibiotic classes. Phylogenetic analysis identified two major ancestral clades. One was genetically diverse with 25 isolates distributed in 16 different sequence types (STs) representing phylogroups A, B1, B2, C and F, while the other comprised phylogroup B2 isolates in subclades representing the ST131, ST73 and ST95 lineages. Forty-seven isolates contained a class 1 integron, of which 14 carried blaCTX -M-gene. Isolates with a class 1 integron carried more antibiotic resistance genes than isolates without an integron and, in most instances, resistance genes were localized within complex resistance loci (CRL). Resistance to fluoroquinolones could be attributed to point mutations in chromosomal parC and gyrB genes and, in addition, two isolates carried a plasmid-associated qnrB4 gene. Co-resistance to fluoroquinolone and broad-spectrum beta-lactam antibiotics was associated with ST131 (HO and CO), ST38 (HO), ST393 (CO), ST2003 (CO) and ST8196 (CO and HO), a novel ST identified in this study. Notably, 10/81 (12.3 %) isolates with ST95 (5 isolates), ST131 (2 isolates), ST88 (2 isolates) and a ST540 likely carry IncFII-IncFIB plasmid replicons with a full spectrum of virulence genes consistent with the carriage of ColV-like plasmids. Our data indicate that IncF plasmids play an important role in shaping virulence and resistance gene carriage in BSI E. coli in Australia.

An Integrated Proteomic and Transcriptomic Analysis Reveals the Venom Complexity of the Bullet Ant Paraponera clavata.

A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.

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.

Evaluation and Diagnosis of the Traumatized Dentition.

Traumatic dental injuries comprise a number of the dental emergency patients who are often seen after hours or on an unscheduled basis in a dental practice environment. Although there are a variety of traumatic dental injuries that can occur, each with their own recommended treatment protocols, the initial evaluation and diagnosis of the traumatized dentition make up a critical aspect of the management of these cases. This article will highlight the key components of a thorough and efficient examination process of the traumatized dentition to include (1) documenting an accurate history of the events causing the injury, (2) performing a systematic clinical examination to include the use of clinical photographs and pulp sensibility tests, (3) obtaining appropriate radiographic images and scans, (4) understanding some considerations unique to evaluating young patients with traumatic injuries, and (5) recognizing the importance of having accurate and thorough documentation of these types of cases. Once the evaluation and diagnosis phase has been completed, the necessary treatment protocols can be initiated in an appropriate manner.

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.

Evaluation and diagnosis of the traumatized dentition.

Traumatic dental injuries comprise a number of the dental emergency patients who are often seen after hours or on an unscheduled basis in a dental practice environment. Although there are a variety of traumatic dental injuries that can occur, each with their own recommended treatment protocols, the initial evaluation and diagnosis of the traumatized dentition make up a critical aspect of the management of these cases. This article will highlight the key components of a thorough and efficient examination process of the traumatized dentition to include (a) documenting an accurate history of the events causing the injury, (b) performing a systematic clinical examination to include the use of clinical photographs and pulp sensibility tests, (c) obtaining appropriate radiographic images and scans, (d) understanding some considerations unique to evaluating young patients with traumatic injuries, and (e) recognizing the importance of having accurate and thorough documentation of these types of cases. Once the evaluation and diagnosis phase has been completed, the necessary treatment protocols can be initiated in an appropriate manner.

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.

A retrospective pilot study to determine whether the reproductive tract microbiota differs between women with a history of infertility and fertile women.

BACKGROUND: We know very little about the microbiota inhabiting the upper female reproductive tract and how it impacts on fertility. AIMS: This pilot study aimed to examine the vaginal, cervical and endometrial microbiota for women with a history of infertility compared to women with a history of fertility. MATERIALS AND METHODS: Using a retrospective case-control study design, women were recruited for collection of vaginal, cervical and endometrial samples. The microbiota composition was analysed by 16S ribosomal RNA (rRNA) gene amplification and endometrial expression of selected human genes by quantitative reverse transcription polymerase chain reaction. RESULTS: Sixty-five specimens from the reproductive tract of 31 women were successfully analysed using 16S rRNA gene amplicon sequencing (16 controls and 15 cases). The dominant microbial community members were consistent in the vagina and cervix, and generally consistent with the endometrium although the relative proportions varied. We detected three major microbiota clusters that did not group by tissue location or case-control status. There was a trend that infertile women more often had Ureaplasma in the vagina and Gardnerella in the cervix. Testing for the expression of selected genes in the endometrium did not show evidence of correlation with case-control status, or with microbial community composition, although Tenascin-C expression correlated with a history of miscarriage. CONCLUSIONS: There is a need for further exploration of the endometrial microbiota, and how the microbiota members or profile interplays with fertility or assisted reproductive technologies.