Publications by authors named "Garry Myers"

60 Publications

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

Sci Rep 2021 May 17;11(1):10399. Epub 2021 May 17.

The iThree Institute, Faculty of Science, University of Technology Sydney, Sydney, Australia.

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.
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http://dx.doi.org/10.1038/s41598-021-89921-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128910PMC
May 2021

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

J Endod 2021 Mar 23;47(3):366-373. Epub 2020 Dec 23.

Department of Endodontics, Virginia Commonwealth University, Richmond, Virginia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.joen.2020.12.011DOI Listing
March 2021

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

Microorganisms 2020 Nov 24;8(12). Epub 2020 Nov 24.

The iThree Institute, University of Technology Sydney, Ultimo 2007, NSW, Australia.

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 , 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 Tn. 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.
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http://dx.doi.org/10.3390/microorganisms8121851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760358PMC
November 2020

Chromatin accessibility dynamics of Chlamydia-infected epithelial cells.

Epigenetics Chromatin 2020 10 27;13(1):45. Epub 2020 Oct 27.

The ithree Institute, University of Technology Sydney, Sydney, NSW, Australia.

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.
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http://dx.doi.org/10.1186/s13072-020-00368-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590614PMC
October 2020

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

J Dent Educ 2021 Mar 12;85(3):383-391. Epub 2020 Oct 12.

Advanced Education Program in Endodontics, Department of Endodontics and Oral Diagnostic Sciences, Virginia Commonwealth University, Richmond, Virginia, USA.

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.
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http://dx.doi.org/10.1002/jdd.12445DOI Listing
March 2021

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

J Endod 2020 Aug 11. Epub 2020 Aug 11.

Department of Endodontics, School of Dentistry, Virginia Commonwealth University, Richmond, Virginia.

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 mm, 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 mm, 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.
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http://dx.doi.org/10.1016/j.joen.2020.07.032DOI Listing
August 2020

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

Emerg Microbes Infect 2020 12;9(1):1780-1792

The ithree institute, University of Technology Sydney, Ultimo, Australia.

The 30Rx subclade of ST131 is a clinically important, globally dispersed pathogenic lineage that typically displays resistance to fluoroquinolones and extended spectrum β-lactams. Isolates EC233 and EC234, variants of ST131-30Rx 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 O25:4A, 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 IS 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 ST131-30Rx clade-C isolates. A MrBayes phylogeny analysis of EC233 and EC234 indicates ST8196 share a most recent common ancestor with ST131-30Rx strain EC70 isolated from the same hospital in 2013. Our study identified genomic hallmarks that define the ST131-30Rx subclade in the ST8196 isolates and highlights a need for unbiased genomic surveillance approaches to identify novel high-risk MDR pathogens that impact healthcare facilities.
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http://dx.doi.org/10.1080/22221751.2020.1797541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473005PMC
December 2020

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

Toxins (Basel) 2020 05 14;12(5). Epub 2020 May 14.

School of Life Sciences, University of Technology Sydney, Broadway, NSW 2007, Australia.

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 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.
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http://dx.doi.org/10.3390/toxins12050324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290781PMC
May 2020

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

Microb Genom 2020 05 6;6(5). Epub 2020 May 6.

The ithree institute, University of Technology Sydney, City Campus, Ultimo, NSW 2007, Australia.

This study sought to assess the genetic variability of isolated from bloodstream infections (BSIs) presenting at Concord Hospital, Sydney during 2013-2016. Whole-genome sequencing was used to characterize 81 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 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 and genes and, in addition, two isolates carried a plasmid-associated 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 in Australia.
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http://dx.doi.org/10.1099/mgen.0.000371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371115PMC
May 2020

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

Front Cell Infect Microbiol 2019 19;9:392. Epub 2019 Nov 19.

Faculty of Science, School of Life Sciences, The ithree Institute, University of Technology Sydney, Ultimo, NSW, Australia.

are Gram-negative obligate intracellular bacterial pathogens responsible for a variety of disease in humans and animals worldwide. 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 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 -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 -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 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.
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http://dx.doi.org/10.3389/fcimb.2019.00392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877545PMC
August 2020

Evaluation and Diagnosis of the Traumatized Dentition.

Authors:
Garry L Myers

J Endod 2019 Dec 14;45(12S):S66-S71. Epub 2019 Oct 14.

Virginia Commonwealth University School of Dentistry, Richmond, Virginia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.joen.2019.05.015DOI Listing
December 2019

Dual RNA-Seq of Chlamydia and Host Cells.

Methods Mol Biol 2019 ;2042:123-135

The iThree Institute, University of Technology Sydney, Ultimo, NSW, Australia.

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.
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http://dx.doi.org/10.1007/978-1-4939-9694-0_9DOI Listing
April 2020

Evaluation and diagnosis of the traumatized dentition.

Authors:
Garry L Myers

Dent Traumatol 2019 Dec 14;35(6):302-308. Epub 2019 Oct 14.

Virginia Commonwealth University School of Dentistry, Richmond, VA, USA.

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.
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http://dx.doi.org/10.1111/edt.12498DOI Listing
December 2019

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

Aust N Z J Obstet Gynaecol 2018 Jun 26;58(3):341-348. Epub 2017 Dec 26.

Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Qld, Australia.

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.
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http://dx.doi.org/10.1111/ajo.12754DOI Listing
June 2018

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

Front Microbiol 2017 21;8:1830. Epub 2017 Sep 21.

School of Life Sciences, The ithree institute, University of Technology SydneyUltimo, NSW, Australia.

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 -infected epithelial cells as proof of principle. Here we provide a detailed laboratory protocol for dual RNA-Seq that is readily adaptable to any host-bacteria system of interest.
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http://dx.doi.org/10.3389/fmicb.2017.01830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613115PMC
September 2017

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

Brief Bioinform 2018 11;19(6):1115-1129

The ithree institute, University of Technology Sydney.

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.
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http://dx.doi.org/10.1093/bib/bbx043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6291798PMC
November 2018

Australian human and parrot Chlamydia psittaci strains cluster within the highly virulent 6BC clade of this important zoonotic pathogen.

Sci Rep 2016 08 4;6:30019. Epub 2016 Aug 4.

Centre for Animal Health Innovation, University of the Sunshine Coast, Sippy Downs, Australia.

Chlamydia psittaci is an avian pathogen and zoonotic agent of atypical pneumonia. The most pathogenic C. psittaci strains cluster into the 6BC clade, predicted to have recently emerged globally. Exposure to infected parrots is a risk factor with limited evidence also of an indirect exposure risk. Genome sequencing was performed on six Australian human and a single avian C. psittaci strain isolated over a 9 year period. Only one of the five human patients had explicit psittacine contact. Genomics analyses revealed that the Australian C. psittaci strains are remarkably similar, clustering tightly within the C. psittaci 6BC clade suggested to have been disseminated by South America parrot importation. Molecular clock analysis using the newly sequenced C. psittaci genomes predicted the emergence of the 6BC clade occurring approximately 2,000 years ago. These findings reveal the potential for an Australian natural reservoir of C. psittaci 6BC strains. These strains can also be isolated from seriously ill patients without explicit psittacine contact. The apparent recent and global spread of C. psittaci 6BC strains raises important questions over how this happened. Further studies may reveal whether the dissemination of this important zoonotic pathogen is linked to Australian parrot importation rather than parrots from elsewhere.
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http://dx.doi.org/10.1038/srep30019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973220PMC
August 2016

The Number, Organization, and Size of Polymorphic Membrane Protein Coding Sequences as well as the Most Conserved Pmp Protein Differ within and across Chlamydia Species.

J Mol Microbiol Biotechnol 2016 28;26(5):333-44. Epub 2016 Jul 28.

Department of Animal Production, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.

Variation is a central trait of the polymorphic membrane protein (Pmp) family. The number of pmp coding sequences differs between Chlamydia species, but it is unknown whether the number of pmp coding sequences is constant within a Chlamydia species. The level of conservation of the Pmp proteins has previously only been determined for Chlamydia trachomatis. As different Pmp proteins might be indispensible for the pathogenesis of different Chlamydia species, this study investigated the conservation of Pmp proteins both within and across C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci. The pmp coding sequences were annotated in 16 C. trachomatis, 6 C. pneumoniae, 2 C. abortus, and 16 C. psittaci genomes. The number and organization of polymorphic membrane coding sequences differed within and across the analyzed Chlamydia species. The length of coding sequences of pmpA,pmpB, and pmpH was conserved among all analyzed genomes, while the length of pmpE/F and pmpG, and remarkably also of the subtype pmpD, differed among the analyzed genomes. PmpD, PmpA, PmpH, and PmpA were the most conserved Pmp in C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci, respectively. PmpB was the most conserved Pmp across the 4 analyzed Chlamydia species.
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http://dx.doi.org/10.1159/000447092DOI Listing
May 2017

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

BMC Genomics 2015 Dec 22;16:1094. Epub 2015 Dec 22.

Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, QLD, Australia.

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.
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http://dx.doi.org/10.1186/s12864-015-2281-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687280PMC
December 2015

Genetic diversity in the plasticity zone and the presence of the chlamydial plasmid differentiates Chlamydia pecorum strains from pigs, sheep, cattle, and koalas.

BMC Genomics 2015 Nov 4;16:893. Epub 2015 Nov 4.

Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Sippy Downs, QLD, 4558, Australia.

Background: Chlamydia pecorum is a globally recognised pathogen of livestock and koalas. To date, comparative genomics of C. pecorum strains from sheep, cattle and koalas has revealed that only single nucleotide polymorphisms (SNPs) and a limited number of pseudogenes appear to contribute to the genetic diversity of this pathogen. No chlamydial plasmid has been detected in these strains despite its ubiquitous presence in almost all other chlamydial species. Genomic analyses have not previously included C. pecorum from porcine hosts. We sequenced the genome of three C. pecorum isolates from pigs with differing pathologies in order to re-evaluate the genetic differences and to update the phylogenetic relationships between C. pecorum from each of the hosts.

Methods: Whole genome sequences for the three porcine C. pecorum isolates (L1, L17 and L71) were acquired using C. pecorum-specific sequence capture probes with culture-independent methods, and assembled in CLC Genomics Workbench. The pairwise comparative genomic analyses of 16 pig, sheep, cattle and koala C. pecorum genomes were performed using several bioinformatics platforms, while the phylogenetic analyses of the core C. pecorum genomes were performed with predicted recombination regions removed. Following the detection of a C. pecorum plasmid, a newly developed C. pecorum-specific plasmid PCR screening assay was used to evaluate the plasmid distribution in 227 C. pecorum samples from pig, sheep, cattle and koala hosts.

Results: Three porcine C. pecorum genomes were sequenced using C. pecorum-specific sequence capture probes with culture-independent methods. Comparative genomics of the newly sequenced porcine C. pecorum genomes revealed an increased average number of SNP differences (~11 500) between porcine and sheep, cattle, and koala C. pecorum strains, compared to previous C. pecorum genome analyses. We also identified a third copy of the chlamydial cytotoxin gene, found only in porcine C. pecorum isolates. Phylogenetic analyses clustered porcine isolates into a distinct clade, highlighting the polyphyletic origin of C. pecorum in livestock. Most surprising, we also discovered a plasmid in the porcine C. pecorum genome. Using this novel C. pecorum plasmid (pCpec) sequence, a) we developed a pCpec screening assay to evaluate the plasmid distribution in C. pecorum from different hosts; and b) to characterise the pCpec sequences from available previously sequenced C. pecorum genome data. pCpec screening showed that the pCpec is common in all hosts of C. pecorum, however not all C. pecorum strains carry pCpec.

Conclusions: This study provides further insight into the complexity of C. pecorum epidemiology and novel genomic regions that may be linked to host specificity. C. pecorum plasmid characterisation may aid in improving our understanding of C. pecorum pathogenesis across the variety of host species this animal pathogen infects.
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http://dx.doi.org/10.1186/s12864-015-2053-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632680PMC
November 2015

Comparative genomic analysis of human Chlamydia pneumoniae isolates from respiratory, brain and cardiac tissues.

Genomics 2015 Dec 28;106(6):373-83. Epub 2015 Sep 28.

Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland, Australia; Faculty of Science, Health, Education & Engineering, University of the Sunshine Coast, Sippy Downs, Queensland, Australia.

Chlamydia pneumoniae is an obligate intracellular bacterium implicated in a wide range of human diseases including atherosclerosis and Alzheimer's disease. Efforts to understand the relationships between C. pneumoniae detected in these diseases have been hindered by the availability of sequence data for non-respiratory strains. In this study, we sequenced the whole genomes for C. pneumoniae isolates from atherosclerosis and Alzheimer's disease, and compared these to previously published C. pneumoniae genomes. Phylogenetic analyses of these new C. pneumoniae strains indicate two sub-groups within human C. pneumoniae, and suggest that both recombination and mutation events have driven the evolution of human C. pneumoniae. Further fine-detailed analyses of these new C. pneumoniae sequences show several genetically variable loci. This suggests that similar strains of C. pneumoniae are found in the brain, lungs and cardiovascular system and that only minor genetic differences may contribute to the adaptation of particular strains in human disease.
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http://dx.doi.org/10.1016/j.ygeno.2015.09.008DOI Listing
December 2015

Chlamydia caviae infection alters abundance but not composition of the guinea pig vaginal microbiota.

Pathog Dis 2015 Jun 10;73(4). Epub 2015 Mar 10.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA

In humans, the vaginal microbiota is thought to be the first line of defense again pathogens including Chlamydia trachomatis. The guinea pig has been extensively used as a model to study chlamydial infection because it shares anatomical and physiological similarities with humans, such as a squamous vaginal epithelium as well as some of the long-term outcomes caused by chlamydial infection. In this study, we aimed to evaluate the guinea pig-C. caviae model of genital infection as a surrogate for studying the role of the vaginal microbiota in the early steps of C. trachomatis infection in humans. We used culture-independent molecular methods to characterize the relative and absolute abundance of bacterial phylotypes in the guinea pig vaginal microbiota in animals non-infected, mock-infected or infected by C. caviae. We showed that the guinea pig and human vaginal microbiotas are of different bacterial composition and abundance. Chlamydia caviae infection had a profound effect on the absolute abundance of bacterial phylotypes but not on the composition of the guinea pig vaginal microbiota. Our findings compromise the validity of the guinea pig-C. caviae model to study the role of the vaginal microbiota during the early steps of sexually transmitted infection.
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http://dx.doi.org/10.1093/femspd/ftv019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445005PMC
June 2015

Culture-independent genome sequencing of clinical samples reveals an unexpected heterogeneity of infections by Chlamydia pecorum.

J Clin Microbiol 2015 May 4;53(5):1573-81. Epub 2015 Mar 4.

Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Sippy Downs, Queensland, Australia

Chlamydia pecorum is an important global pathogen of livestock, and it is also a significant threat to the long-term survival of Australia's koala populations. This study employed a culture-independent DNA capture approach to sequence C. pecorum genomes directly from clinical swab samples collected from koalas with chlamydial disease as well as from sheep with arthritis and conjunctivitis. Investigations into single-nucleotide polymorphisms within each of the swab samples revealed that a portion of the reads in each sample belonged to separate C. pecorum strains, suggesting that all of the clinical samples analyzed contained mixed populations of genetically distinct C. pecorum isolates. This observation was independent of the anatomical site sampled and the host species. Using the genomes of strains identified in each of these samples, whole-genome phylogenetic analysis revealed that a clade containing a bovine and a koala isolate is distinct from other clades comprised of livestock or koala C. pecorum strains. Providing additional evidence to support exposure of koalas to Australian livestock strains, two minor strains assembled from the koala swab samples clustered with livestock strains rather than koala strains. Culture-independent probe-based genome capture and sequencing of clinical samples provides the strongest evidence yet to suggest that naturally occurring chlamydial infections are comprised of multiple genetically distinct strains.
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http://dx.doi.org/10.1128/JCM.03534-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4400774PMC
May 2015

Comparative genomics of koala, cattle and sheep strains of Chlamydia pecorum.

BMC Genomics 2014 Aug 8;15:667. Epub 2014 Aug 8.

Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Sippy Downs 4558, Queensland, Australia.

Background: Chlamydia pecorum is an important pathogen of domesticated livestock including sheep, cattle and pigs. This pathogen is also a key factor in the decline of the koala in Australia. We sequenced the genomes of three koala C. pecorum strains, isolated from the urogenital tracts and conjunctiva of diseased koalas. The genome of the C. pecorum VR629 (IPA) strain, isolated from a sheep with polyarthritis, was also sequenced.

Results: Comparisons of the draft C. pecorum genomes against the complete genomes of livestock C. pecorum isolates revealed that these strains have a conserved gene content and order, sharing a nucleotide sequence similarity > 98%. Single nucleotide polymorphisms (SNPs) appear to be key factors in understanding the adaptive process. Two regions of the chromosome were found to be accumulating a large number of SNPs within the koala strains. These regions include the Chlamydia plasticity zone, which contains two cytotoxin genes (toxA and toxB), and a 77 kbp region that codes for putative type III effector proteins. In one koala strain (MC/MarsBar), the toxB gene was truncated by a premature stop codon but is full-length in IPTaLE and DBDeUG. Another five pseudogenes were also identified, two unique to the urogenital strains C. pecorum MC/MarsBar and C. pecorum DBDeUG, respectively, while three were unique to the koala C. pecorum conjunctival isolate IPTaLE. An examination of the distribution of these pseudogenes in C. pecorum strains from a variety of koala populations, alongside a number of sheep and cattle C. pecorum positive samples from Australian livestock, confirmed the presence of four predicted pseudogenes in koala C. pecorum clinical samples. Consistent with our genomics analyses, none of these pseudogenes were observed in the livestock C. pecorum samples examined. Interestingly, three SNPs resulting in pseudogenes identified in the IPTaLE isolate were not found in any other C. pecorum strain analysed, raising questions over the origin of these point mutations.

Conclusions: The genomic data revealed that variation between C. pecorum strains were mainly due to the accumulation of SNPs, some of which cause gene inactivation. The identification of these genetic differences will provide the basis for further studies to understand the biology and evolution of this important animal pathogen.
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http://dx.doi.org/10.1186/1471-2164-15-667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4137089PMC
August 2014

Circleator: flexible circular visualization of genome-associated data with BioPerl and SVG.

Bioinformatics 2014 Nov 29;30(21):3125-7. Epub 2014 Jul 29.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, i3 Institute, University of Technology, Sydney, PO Box 123 Broadway NSW 2007, Australia, Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, MD 21201, Center for Health-Related Informatics and Bioimaging, University of Maryland, College Park, MD 20740 and Department of Epidemiology and Public Health, University of Maryland School of Medicine, Baltimore, MD 21201, USA Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, i3 Institute, University of Technology, Sydney, PO Box 123 Broadway NSW 2007, Australia, Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, MD 21201, Center for Health-Related Informatics and Bioimaging, University of Maryland, College Park, MD 20740 and Department of Epidemiology and Public Health, University of Maryland School of Medicine, Baltimore, MD 21201, USA Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, i3 Institute, University of Technology, Sydney, PO Box 123 Broadway NSW 2007, Australia, Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, MD 21201, Center for Health-Related Informatics and Bioimaging, University of Maryland, College Park, MD 20740 and Department of Epidemiology and Public Health, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

Summary: Circleator is a Perl application that generates circular figures of genome-associated data. It leverages BioPerl to support standard annotation and sequence file formats and produces publication-quality SVG output. It is designed to be both flexible and easy to use. It includes a library of circular track types and predefined configuration files for common use-cases, including. (i) visualizing gene annotation and DNA sequence data from a GenBank flat file, (ii) displaying patterns of gene conservation in related microbial strains, (iii) showing Single Nucleotide Polymorphisms (SNPs) and indels relative to a reference genome and gene set and (iv) viewing RNA-Seq plots.

Availability And Implementation: Circleator is freely available under the Artistic License 2.0 from http://jonathancrabtree.github.io/Circleator/ and is integrated with the CloVR cloud-based sequence analysis Virtual Machine (VM), which can be downloaded from http://clovr.org or run on Amazon EC2.
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http://dx.doi.org/10.1093/bioinformatics/btu505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201160PMC
November 2014

Early microRNA expression profile as a prognostic biomarker for the development of pelvic inflammatory disease in a mouse model of chlamydial genital infection.

mBio 2014 Jun 24;5(3):e01241-14. Epub 2014 Jun 24.

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences and Arkansas Children's Hospital Research Institute, Little Rock, Arkansas, USA.

Unlabelled: It is not currently possible to predict the probability of whether a woman with a chlamydial genital infection will develop pelvic inflammatory disease (PID). To determine if specific biomarkers may be associated with distinct chlamydial pathotypes, we utilized two Chlamydia muridarum variants (C. muridarum Var001 [CmVar001] and CmVar004) that differ in their abilities to elicit upper genital tract pathology in a mouse model. CmVar004 has a lower growth rate in vitro and induces pathology in only 20% of C57BL/6 mouse oviducts versus 83.3% of oviducts in CmVar001-infected mice. To determine if chemokine and cytokine production within 24 h of infection is associated with the outcome of pathology, levels of 15 chemokines and cytokines were measured. CmVar004 infection induced significantly lower levels of CXCL1, CXCL2, tumor necrosis factor alpha (TNF-α), and CCL2 in comparison to CmVar001 infection with similar rRNA (rs16) levels for Chlamydiae. A combination of microRNA (miRNA) sequencing and quantitative real-time PCR (qRT-PCR) analysis of 134 inflammation-related miRNAs was performed 24 h postinfection to determine if the chemokine/cytokine responses would also be reflected in miRNA expression profiles. Interestingly, 12 miRNAs (miR-135a-5p, miR298-5p, miR142-3p, miR223-3p, miR299a-3p, miR147-3p, miR105, miR325-3p, miR132-3p, miR142-5p, miR155-5p, and miR-410-3p) were overexpressed during CmVar004 infection compared to CmVar001 infection, inversely correlating with the respective chemokine/cytokine responses. To our knowledge, this is the first report demonstrating that early biomarkers elicited in the host can differentiate between two pathological variants of chlamydiae and be predictive of upper tract disease.

Importance: It is apparent that an infecting chlamydial population consists of multiple genetic variants with differing capabilities of eliciting a pathological response; thus, it may be possible to identify biomarkers specific for a given virulence pathotype. miRNAs are known to regulate genes that in turn regulate signaling pathways involved in disease pathogenesis. Importantly, miRNAs are stable and can reflect a tissue response and therefore have the potential to be biomarkers of disease severity. Currently, with respect to chlamydial infections, there is no way to predict whether an infected patient is more or less likely to develop PID. However, data presented in this study indicate that the expression of a specific miRNA profile associated with a virulent variant early in the infection course may be predictive of an increased risk of pelvic inflammatory disease, allowing more aggressive treatment before significant pathology develops.
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http://dx.doi.org/10.1128/mBio.01241-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073489PMC
June 2014

Standardized metadata for human pathogen/vector genomic sequences.

PLoS One 2014 17;9(6):e99979. Epub 2014 Jun 17.

Broad Institute, Cambridge, Massachusetts, United States of America.

High throughput sequencing has accelerated the determination of genome sequences for thousands of human infectious disease pathogens and dozens of their vectors. The scale and scope of these data are enabling genotype-phenotype association studies to identify genetic determinants of pathogen virulence and drug/insecticide resistance, and phylogenetic studies to track the origin and spread of disease outbreaks. To maximize the utility of genomic sequences for these purposes, it is essential that metadata about the pathogen/vector isolate characteristics be collected and made available in organized, clear, and consistent formats. Here we report the development of the GSCID/BRC Project and Sample Application Standard, developed by representatives of the Genome Sequencing Centers for Infectious Diseases (GSCIDs), the Bioinformatics Resource Centers (BRCs) for Infectious Diseases, and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), informed by interactions with numerous collaborating scientists. It includes mapping to terms from other data standards initiatives, including the Genomic Standards Consortium's minimal information (MIxS) and NCBI's BioSample/BioProjects checklists and the Ontology for Biomedical Investigations (OBI). The standard includes data fields about characteristics of the organism or environmental source of the specimen, spatial-temporal information about the specimen isolation event, phenotypic characteristics of the pathogen/vector isolated, and project leadership and support. By modeling metadata fields into an ontology-based semantic framework and reusing existing ontologies and minimum information checklists, the application standard can be extended to support additional project-specific data fields and integrated with other data represented with comparable standards. The use of this metadata standard by all ongoing and future GSCID sequencing projects will provide a consistent representation of these data in the BRC resources and other repositories that leverage these data, allowing investigators to identify relevant genomic sequences and perform comparative genomics analyses that are both statistically meaningful and biologically relevant.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099979PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4061050PMC
February 2015

Genome Sequence of Chlamydia suis MD56, Isolated from the Conjunctiva of a Weaned Piglet.

Genome Announc 2014 May 8;2(3). Epub 2014 May 8.

DIMES, Section of Microbiology, University of Bologna, Bologna, Italy.

Chlamydia suis is a natural pathogen of pigs (Sus scrofa) and causes conjunctivitis, pneumonia, enteritis, and various reproductive disorders that adversely impact this economically important animal. Here, we report the first C. suis genome, that of C. suis MD56, isolated from a conjunctival swab of a weaned piglet.
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http://dx.doi.org/10.1128/genomeA.00425-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014695PMC
May 2014

Evidence for the existence of two new members of the family Chlamydiaceae and proposal of Chlamydia avium sp. nov. and Chlamydia gallinacea sp. nov.

Syst Appl Microbiol 2014 Mar 22;37(2):79-88. Epub 2014 Jan 22.

Faculty of Mathematics and Computer Science, Friedrich-Schiller-Universität, Jena, Germany.

The family Chlamydiaceae with the recombined single genus Chlamydia currently comprises nine species, all of which are obligate intracellular organisms distinguished by a unique biphasic developmental cycle. Anecdotal evidence from epidemiological surveys in flocks of poultry, pigeons and psittacine birds have indicated the presence of non-classified chlamydial strains, some of which may act as pathogens. In the present study, phylogenetic analysis of ribosomal RNA and ompA genes, as well as multi-locus sequence analysis of 11 field isolates were conducted. All independent analyses assigned the strains into two different clades of monophyletic origin corresponding to pigeon and psittacine strains or poultry isolates, respectively. Comparative genome analysis involving the type strains of currently accepted Chlamydiaceae species and the designated type strains representing the two new clades confirmed that the latter could be classified into two different species as their average nucleotide identity (ANI) values were always below 94%, both with the closest relative species and between themselves. In view of the evidence obtained from the analyses, we propose the addition of two new species to the current classification: Chlamydia avium sp. nov. comprising strains from pigeons and psittacine birds (type strain 10DC88(T); DSMZ: DSM27005(T), CSUR: P3508(T)) and Chlamydia gallinacea sp. nov. comprising strains from poultry (type strain 08-1274/3(T); DSMZ: DSM27451(T), CSUR: P3509(T)).
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http://dx.doi.org/10.1016/j.syapm.2013.12.004DOI Listing
March 2014

Genome sequencing and comparative analysis of three Chlamydia pecorum strains associated with different pathogenic outcomes.

BMC Genomics 2014 Jan 14;15:23. Epub 2014 Jan 14.

Moredun Research Institute, Pentlands Science Park, Bush Loan, Edinburgh, Midlothian EH26 0PZ, UK.

Background: Chlamydia pecorum is the causative agent of a number of acute diseases, but most often causes persistent, subclinical infection in ruminants, swine and birds. In this study, the genome sequences of three C. pecorum strains isolated from the faeces of a sheep with inapparent enteric infection (strain W73), from the synovial fluid of a sheep with polyarthritis (strain P787) and from a cervical swab taken from a cow with metritis (strain PV3056/3) were determined using Illumina/Solexa and Roche 454 genome sequencing.

Results: Gene order and synteny was almost identical between C. pecorum strains and C. psittaci. Differences between C. pecorum and other chlamydiae occurred at a number of loci, including the plasticity zone, which contained a MAC/perforin domain protein, two copies of a >3400 amino acid putative cytotoxin gene and four (PV3056/3) or five (P787 and W73) genes encoding phospholipase D. Chlamydia pecorum contains an almost intact tryptophan biosynthesis operon encoding trpABCDFR and has the ability to sequester kynurenine from its host, however it lacks the genes folA, folKP and folB required for folate metabolism found in other chlamydiae. A total of 15 polymorphic membrane proteins were identified, belonging to six pmp families. Strains possess an intact type III secretion system composed of 18 structural genes and accessory proteins, however a number of putative inc effector proteins widely distributed in chlamydiae are absent from C. pecorum. Two genes encoding the hypothetical protein ORF663 and IncA contain variable numbers of repeat sequences that could be associated with persistence of infection.

Conclusions: Genome sequencing of three C. pecorum strains, originating from animals with different disease manifestations, has identified differences in ORF663 and pseudogene content between strains and has identified genes and metabolic traits that may influence intracellular survival, pathogenicity and evasion of the host immune system.
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http://dx.doi.org/10.1186/1471-2164-15-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3932018PMC
January 2014
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