Bartonella spp. Infections Identified by Molecular Methods, United States.

Molecular methods can enable rapid identification of Bartonella spp. infections, which are difficult to diagnose by using culture or serology. We analyzed clinical test results of PCR that targeted bacterial 16S rRNA hypervariable V1-V2 regions only or in parallel with PCR of Bartonella-specific ribC gene. We identified 430 clinical specimens infected with Bartonella spp. from 420 patients in the United States. Median patient age was 37 (range 1-79) years; 62% were male. We identified B. henselae in 77%, B. quintana in 13%, B. clarridgeiae in 1%, B. vinsonii in 1%, and B. washoensis in 1% of specimens. B. quintana was detected in 83% of cardiac specimens; B. henselae was detected in 34% of lymph node specimens. We detected novel or uncommon Bartonella spp. in 9 patients. Molecular diagnostic testing can identify Bartonella spp. infections, including uncommon and undescribed species, and might be particularly useful for patients who have culture-negative endocarditis or lymphadenitis.

Sensitive Identification of Bacterial DNA in Clinical Specimens by Broad-Range 16S rRNA Gene Enrichment.

The broad-range detection and identification of bacterial DNA from clinical specimens are a foundational approach in the practice of molecular microbiology. However, there are circumstances under which conventional testing may yield false-negative or otherwise uninterpretable results, including the presence of multiple bacterial templates or degraded nucleic acids. Here, we describe an alternative, next-generation sequencing approach for the broad range detection of bacterial DNA using broad-range 16S rRNA gene hybrid capture ("16S Capture"). The method is able to deconvolute multiple bacterial species present in a specimen, is compatible with highly fragmented templates, and can be readily implemented when the overwhelming majority of nucleic acids in a specimen derive from the human host. We find that this approach is sensitive to detecting as few as 17 Staphylococcus aureus genomes from a background of 100 ng of human DNA, providing 19- to 189-fold greater sensitivity for identifying bacterial sequences than standard shotgun metagenomic sequencing, and is able to successfully recover organisms from across the eubacterial tree of life. Application of 16S Capture to a proof-of-principle case series demonstrated its ability to identify bacterial species that were consistent with histological evidence of infection, even when diagnosis could not be established using conventional broad range bacterial detection assays. 16S Capture provides a novel means for the efficient and sensitive detection of bacteria embedded in human tissues and for specimens containing highly fragmented template DNA.

Cell free DNA from respiratory pathogens is detectable in the blood plasma of Cystic Fibrosis patients.

Diagnostically informative microbial cell-free DNA (cfDNA) can be detected from blood plasma during fulminant infections such as sepsis. However, the potential for DNA from airway pathogens to enter the circulation of cystic fibrosis (CF) patients during chronic infective states has not yet been evaluated. We assessed whether patient blood contained measurable quantities of cfDNA from CF respiratory microorganisms by sequencing plasma from 21 individuals with CF recruited from outpatient clinics and 12 healthy controls. To account for possible contamination with exogenous microbial nucleic acids, statistical significance of microbe-derived read counts from CF patients was determined relative to the healthy control population. In aggregate, relative abundance of microbial cfDNA was nearly an order of magnitude higher in CF patients than in healthy subjects (p = 8.0×10-3). 15 of 21 (71%) CF patients demonstrated cfDNA from one or more relevant organisms. In contrast, none of the healthy subjects evidenced significant microbial cfDNA for any of the organisms examined. Concordance of cfDNA with standard microbiological culture of contemporaneously collected patient sputum was variable. Our findings provide evidence that cfDNA from respiratory pathogens are present in the bloodstream of most CF patients, which could potentially be exploited for the purposes of noninvasive clinical diagnosis.

Comprehensive evaluation of complex polymicrobial specimens using next generation sequencing and standard microbiological culture.

Optimal clinical decision-making depends on identification of clinically relevant organisms present in a sample. Standard microbiological culture may fail to identify unusual or fastidious organisms and can misrepresent relative abundance of sample constituents. Culture-independent methods have improved our ability to deconvolute polymicrobial patient samples. We used next-generation 16S rRNA gene sequencing (NGS16S) to determine how often cultivatable organisms in complex polymicrobial samples are not reported by standard culture. Twenty consecutive bronchoalveolar lavage (BAL) samples were plated to standard and additional media; bacteria were identified by NGS16S analysis of DNA extracted directly from samples or from washed culture plates. 96% of organisms identified were cultivable, but only 21% were reported by standard culture, indicating that standard work-up provides an incomplete assessment of microbial constituents. Direct NGS16S correlated well with standard culture, identifying the same predominant organism in 50% of samples. When predominant organisms differed, NGS16S most often detected anaerobes, whose growth is unsupported by standard culture conditions for this specimen. NGS16S identified more organisms per sample and allowed identification of fastidious organisms, while culture was better at capturing organisms when bacterial load was low, and allowed incidental recovery of non-bacterial pathogens. Molecular and culture-based methods together detect more organisms than either method alone.

Patterns of care for stage III non-small cell lung cancer in Australia.

Stage III non-small cell lung cancer (NSCLC) makes up a third of all NSCLC cases and is potentially curable. Despite this 5-year survival rates remain between 15% and 20% with chemoradiation treatment alone given with curative intent. With the recent exciting breakthroughs in immunotherapy use (durvalumab) for stage III NSCLC, further improvements in patient survival can be expected. Most patients with stage III NSCLC present initially to their general practitioner (GP). The recommended time from GP referral to first specialist appointment is less than 14 days with treatment initiated within 42 days. Our review found that there is a shortfall in meeting these recommendations, however a number of initiatives have been established in Australia to improve timely and accurate diagnosis and treatment patterns. The lung cancer multidisciplinary team (MDT) is critical to consistency of evidence-based diagnosis and treatment and can improve patient survival. We aimed to review current patterns of care and clinical practice recommendations for stage III NSCLC across Australia and identify opportunities to improve practice in referral, diagnosis and treatment pathways.

The role of omega-3 polyunsaturated fatty acids in cardiovascular health.

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) play an important role in both primary and secondary prevention of cardiovascular diseases (CVDs). Clinicians should strongly consider therapy with ω-3 PUFAs for individuals at risk for CVD and with known CVD, particularly individuals who are at increased risk for sudden cardiac death. The amount of ω-3 PUFA in erythrocyte membranes, which is the ω-3 index, could prove to be a modifiable risk factor for CVD. This review outlines potential mechanisms of action, the ω-3 index, results of clinical and prevention trials, and recommendations for ω-3 PUFA therapy in CVD. The majority of epidemiologic and controlled interventional studies have demonstrated that marine- and plant-derived ω-3 PUFAs have beneficial effects on CVD. The ω-3 index may prove to be a valuable tool for assessing an individual's need for increased ω-3 PUFAs through diet or supplementation.

Comparison of cardiomyogenic potential among human ESC and iPSC lines.

We recently reported that, following induction of clumps of pluripotent H1 human embryonic stem cells (hESCs) with activin-A and Bmp4 in defined medium for 5 days, widespread differentiation of rhythmically contracting cardiomyocytes occurs within 3-4 weeks. In this study, the same approach was used to assess whether human induced pluripotent stem cells (hiPSCs), which may theoretically provide an unlimited source of patient-matched cells for transplantation therapy, can similarly undergo cardiomyocyte differentiation. Differentiation of four pluripotent cell lines (H1 and H9 hESCs and C2a and C6a hiPSCs) was compared in parallel by monitoring rhythmic contraction, morphologic differentiation, and expression of cardiomyogenic genes. Based on expression of the cardiomyogenic lineage markers MESP1, ISL1, and NKX2-5, all four cell lines were induced into the cardiomyogenic lineage. However, in contrast to the widespread appearance of striations and rhythmic contractility seen in H9 and especially in H1 hESCs, both hiPSC lines exhibited poor terminal differentiation. These findings suggest that refined modes of generating hiPSCs, as well as of inducing cardiomyogenesis in them, may be required to fulfill their potential as agents of cardiac regeneration.

Who benefits from emotional expression? An examination of personality differences among gynaecological cancer patients participating in a randomized controlled emotional disclosure intervention trial.

The present study examined the role of neuroticism and extraversion in the effects of written emotional disclosure in patients diagnosed with gynaecological cancer. It was hypothesized that high levels of neuroticism would be associated with an increase in distress after emotional disclosure as mediated by heightened negative affect and avoidance post-disclosure. Conversely, we expected high extraversion to be associated with decreased distress as mediated by heightened positive moods and a decrease in avoidance. Eighty-eight participants were randomly assigned to participate in an expressive writing task versus a control writing task. Distress and avoidance were assessed at baseline and 6 months post-writing. Negative and positive mood were assessed immediately following writing. Multiple regression confirmed that neuroticism but not extraversion moderates the effects of emotional disclosure on distress, however no significant mediating relationships were found.

Biogenesis of bacterial membrane vesicles.

Membrane vesicle (MV) release remains undefined, despite its conservation among replicating Gram-negative bacteria both in vitro and in vivo. Proteins identified in Salmonella MVs, derived from the envelope, control MV production via specific defined domains that promote outer membrane protein-peptidoglycan (OM-PG) and OM protein-inner membrane protein (OM-PG-IM) interactions within the envelope structure. Modulation of OM-PG and OM-PG-IM interactions along the cell body and at division septa, respectively, maintains membrane integrity while co-ordinating localized release of MVs with distinct size distribution and protein content. These data support a model of MV biogenesis, wherein bacterial growth and division invoke temporary, localized reductions in the density of OM-PG and OM-PG-IM associations within the envelope structure, thus releasing OM as MVs.

Salmonella typhimurium coordinately regulates FliC location and reduces dendritic cell activation and antigen presentation to CD4+ T cells.

During infection, Salmonella transitions from an extracellular-phase (STEX, growth outside host cells) to an intracellular-phase (STIN, growth inside host cells): changes in gene expression mediate survival in the phagosome and modifies LPS and outer membrane protein expression, including altered production of FliC, an Ag recognized by immune CD4+ T cells. Previously, we demonstrated that systemic STIN bacteria repress FliC below the activation threshold of FliC-specific T cells. In this study, we tested the hypothesis that changes in FliC compartmentalization and bacterial responses triggered during the transition from STEX to STIN combine to reduce the ability of APCs to present FliC to CD4+ T cells. Approximately 50% of the Salmonella-specific CD4+ T cells from Salmonella-immune mice were FliC specific and produced IFN-gamma, demonstrating the potent immunogenicity of FliC. FliC expressed by STEX bacteria was efficiently presented by splenic APCs to FliC-specific CD4+ T cells in vitro. However, STIN bacteria, except when lysed, expressed FliC within a protected intracellular compartment and evaded stimulation of FliC-specific T cells. The combination of STIN-mediated responses that reduced FliC bioavailability were overcome by dendritic cells (DCs), which presented intracellular FliC within heat-killed bacteria; however, this ability was abrogated by live bacterial infection. Furthermore, STIN bacteria, unlike STEX, limited DC activation as measured by increased MHC class II, CD86, TNF-alpha, and IL-12 expression. These data indicate that STIN bacteria restrict FliC bioavailability by Ag compartmentalization, and together with STIN bacterial responses, limit DC maturation and cytokine production. Together, these mechanisms may restrain DC-mediated activation of FliC-specific CD4+ T cells.

Evasion of Toll-like receptor 5 by flagellated bacteria.

Toll-like receptor 5 (TLR5) recognizes an evolutionarily conserved site on bacterial flagellin that is required for flagellar filament assembly and motility. The alpha and epsilon Proteobacteria, including the important human pathogens Campylobacter jejuni, Helicobacter pylori, and Bartonella bacilliformis, require flagellar motility to efficiently infect mammalian hosts. In this study, we demonstrate that these bacteria make flagellin molecules that are not recognized by TLR5. We map the site responsible for TLR5 evasion to amino acids 89-96 of the N-terminal D1 domain, which is centrally positioned within the previously defined TLR5 recognition site. Salmonella flagellin is strongly recognized by TLR5, but mutating residues 89-96 to the corresponding H. pylori flaA sequence abolishes TLR5 recognition and also destroys bacterial motility. To preserve bacterial motility, alpha and epsilon Proteobacteria possess compensatory amino acid changes in other regions of the flagellin molecule, and we engineer a mutant form of Salmonella flagellin that evades TLR5 but retains motility. These results suggest that TLR5 evasion is critical for the survival of this subset of bacteria at mucosal sites in animals and raise the intriguing possibility that flagellin receptors provided the selective force to drive the evolution of these unique subclasses of bacterial flagellins.

FliC-specific CD4+ T cell responses are restricted by bacterial regulation of antigen expression.

Salmonella typhimurium, a facultatively intracellular pathogen, regulates expression of virulence factors in response to distinct environments encountered during the course of infection. We tested the hypothesis that the transition from extra- to intracellular environments during Salmonella infection triggers changes in Ag expression that impose both temporal and spatial limitations on the host T cell response. CD4(+) T cells recovered from Salmonella immune mice were propagated in vitro using Ag derived from bacteria grown in conditions designed to emulate extra- or intracellular environments in vivo. Extracellular phase bacteria supported a dominant T cell response to the flagellar subunit protein FliC, whereas intracellular phase bacteria were unable to support expansion of FliC-specific T cells from populations known to contain T cells with reactivity to this Ag. This result was attributed to bacterial regulation of FliC expression: transcription and protein levels were repressed in bacteria growing in the spleens of infected mice. Furthermore, Salmonella-infected splenocytes taken directly ex vivo stimulated FliC-specific T cell clones only when intracellular FliC expression was artificially up-regulated. Although it has been suggested that a microanatomical separation of immune T cells and infected APC exists in vivo, we demonstrate that intracellular Salmonella can repress FliC expression below the T cell activation threshold. This potentially provides a mechanism for intracellular Salmonella at systemic sites to avoid detection by Ag-specific T cells primed at intestinal sites early in infection.

CD4+ T cells and toll-like receptors recognize Salmonella antigens expressed in bacterial surface organelles.

A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysaccharide (LPS). PhoP/PhoQ, a regulon controlling Salmonella virulence and remodeling of LPS to resist innate immunity, coordinately represses production of surface-exposed antigens recognized by CD4+ T cells and TLRs. These data suggest that genetically coordinated surface modifications may provide a growth advantage for Salmonella in host tissues by limiting both innate and adaptive immune recognition.

Characterization of a novel group of mycobacteria and proposal of Mycobacterium sherrisii sp. nov.

We describe here the characterization of five isolates of Mycobacterium simiae-like organisms representing a novel group based on whole-cell fatty acid analysis and genotypic evaluation. Two of the five isolates in this study, W55 and W58, were previously considered to belong to M. simiae serotype 2. Analysis of cellular fatty acids by gas-liquid chromatography indicated a close clustering of this group, which was well differentiated from the other M. simiae-like species. Molecular characterization was performed by nucleic acid sequencing of the small subunit rRNA gene and the gene encoding the 65-kDa heat shock protein and genomic DNA hybridization. Sequence analysis of the entire 16S rRNA gene showed a unique sequence most closely related to those of M. triplex and M. simiae. The hsp65 partial gene sequence was identical for the five isolates, with 97% identity to the M. simiae type strain. However, qualitative whole genomic DNA hybridization analysis confirmed that this group is genetically distinct from M. simiae and M. triplex. Antimicrobial susceptibilities for this group resemble those of M. simiae and M. lentiflavum. We conclude that this group represents a unique Mycobacterium species for which we propose the name Mycobacterium sherrisii sp. nov.

Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility.

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and activates host inflammatory responses. In this study, we examine the nature of the TLR5-flagellin interaction. With deletional, insertional and alanine-scanning mutagenesis, we precisely mapped the TLR5 recognition site on flagellin to a cluster of 13 amino acid residues that participate in intermolecular interactions within flagellar protofilaments and that are required for bacterial motility. The recognition site is buried in the flagellar filament, and monomeric flagellin, but not the filamentous molecule, stimulated TLR5. Finally, flagellin coprecipitated with TLR5, indicating close physical interaction between the molecules. These studies demonstrate the exquisite ability of the innate immune system to precisely target a conserved site on flagellin that is essential for bacterial motility.