Characterization of an experimental model to determine streptococcal M protein-induced autoimmune cardiac and neurobehavioral abnormalities.

Group A streptococcal (GAS) infection is associated with a spectrum of autoimmune diseases including acute rheumatic fever/rheumatic heart disease (ARF/RHD) and neurobehavioral abnormalities. Antibodies against GAS M proteins cross-react with host tissue proteins in the heart and brain leading to the symptomatology observed in ARF/RHD. As throat carriage of Streptococcus dysgalactiae subspecies equisimilis (SDSE) has been reported to be relatively high in some ARF/RHD endemic regions compared with GAS, and both SDSE and GAS express coiled-coil surface protein called M protein, we hypothesized that streptococci other than GAS can also associated with ARF/RHD and neurobehavioral abnormalities. Neurobehavioral assessments and electrocardiography were performed on Lewis rats before and after exposure to recombinant GAS and SDSE M proteins. Histological assessments were performed to confirm inflammatory changes in cardiac and neuronal tissues. ELISA and Western blot analysis were performed to determine the cross-reactivity of antibodies with host connective, cardiac and neuronal tissue proteins. Lewis rats injected with M proteins either from GAS or SDSE developed significant cardiac functional and neurobehavioral abnormalities in comparison to control rats injected with phosphate-buffered saline. Antibodies against GAS and SDSE M proteins cross-reacted with cardiac, connective and neuronal proteins. Serum from rats injected with streptococcal antigens showed higher immunoglobulin G binding to the striatum and cortex of the brain. Cardiac and neurobehavioral abnormalities observed in our experimental model were comparable to the cardinal symptoms observed in patients with ARF/RHD. Here for the first time, we demonstrate in an experimental model that M proteins from different streptococcal species could initiate and drive the autoimmune-mediated cardiac tissue damage and neurobehavioral abnormalities.

Dressings and securements for the prevention of peripheral intravenous catheter failure in adults (SAVE): a pragmatic, randomised controlled, superiority trial.

BACKGROUND: Two billion peripheral intravenous catheters (PIVCs) are used globally each year, but optimal dressing and securement methods are not well established. We aimed to compare the efficacy and costs of three alternative approaches to standard non-bordered polyurethane dressings. METHODS: We did a pragmatic, randomised controlled, parallel-group superiority trial at two hospitals in Queensland, Australia. Eligible patients were aged 18 years or older and required PIVC insertion for clinical treatment, which was expected to be required for longer than 24 h. Patients were randomly assigned (1:1:1:1) via a centralised web-based randomisation service using random block sizes, stratified by hospital, to receive tissue adhesive with polyurethane dressing, bordered polyurethane dressing, a securement device with polyurethane dressing, or polyurethane dressing (control). Randomisation was concealed before allocation. Patients, clinicians, and research staff were not masked because of the nature of the intervention, but infections were adjudicated by a physician who was masked to treatment allocation. The primary outcome was all-cause PIVC failure (as a composite of complete dislodgement, occlusion, phlebitis, and infection [primary bloodstream infection or local infection]). Analysis was by modified intention to treat. This trial is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12611000769987. FINDINGS: Between March 18, 2013, and Sept 9, 2014, we randomly assigned 1807 patients to receive tissue adhesive with polyurethane (n=446), bordered polyurethane (n=454), securement device with polyurethane (n=453), or polyurethane (n=454); 1697 patients comprised the modified intention-to-treat population. 163 (38%) of 427 patients in the tissue adhesive with polyurethane group (absolute risk difference -4·5% [95% CI -11·1 to 2·1%], p=0·19), 169 (40%) of 423 of patients in the bordered polyurethane group (-2·7% [-9·3 to 3·9%] p=0·44), 176 (41%) of 425 patients in the securement device with poplyurethane group (-1·2% [-7·9% to 5·4%], p=0·73), and 180 (43%) of 422 patients in the polyurethane group had PIVC failure. 17 patients in the tissue adhesive with polyurethane group, two patients in the bordered polyurethane group, eight patients in the securement device with polyurethane group, and seven patients in the polyurethane group had skin adverse events. Total costs of the trial interventions did not differ significantly between groups. INTERPRETATION: Current dressing and securement methods are commonly associated with PIVC failure and poor durability, with simultaneous use of multiple products commonly required. Cost is currently the main factor that determines product choice. Innovations to achieve effective, durable dressings and securements, and randomised controlled trials assessing their effectiveness are urgently needed. FUNDING: Australian National Health and Medical Research Council.

Antibacterial 5α-Spirostane Saponins from the Fruit of Cordyline manners-suttoniae.

Antibacterial-activity-guided fractionation of a dichloromethane extract from the fruit of Cordyline manners-suttoniae and subsequent structure-activity investigations resulted in the identification of 10 new (1-10) and one known (11) 5α-spirostane saponin. The structures of the new compounds were established by 1D and 2D NMR analyses. The absolute configurations of the isolated compounds were determined by X-ray diffraction analysis or chemical derivatizations. The most active compound, suttonigenin F (6), inhibited the Gram-positive bacteria Staphylococcus aureus with MIC75 values that were comparable to those of the antibiotic chloramphenicol. Structure-activity relationships were also obtained from the assessment of antibacterial and cytotoxic activities of the isolated saponins.

Group G Streptococcus Induces an Autoimmune Carditis Mediated by Interleukin 17A and Interferon γ in the Lewis Rat Model of Rheumatic Heart Disease.

Acute rheumatic fever and rheumatic heart disease (ARF/RHD) have long been described as autoimmune sequelae of Streptococcus pyogenes or group A streptococcal (GAS) infection. Both antibody and T-cell responses against immunodominant GAS virulence factors, including M protein, cross-react with host tissue proteins, triggering an inflammatory response leading to permanent heart damage. However, in some ARF/RHD-endemic regions, throat carriage of GAS is low. Because Streptococcus dysgalactiae subspecies equisimilis organisms, also known as ß-hemolytic group C streptococci and group G streptococci (GGS), also express M protein, we postulated that streptococci other than GAS may have the potential to initiate or exacerbate ARF/RHD. Using a model initially developed to investigate the uniquely human disease of ARF/RHD, we have discovered that GGS causes interleukin 17A/interferon γ-induced myocarditis and valvulitis, hallmarks of ARF/RHD. Remarkably the histological, immunological, and functional changes in the hearts of rats exposed to GGS are identical to those exposed to GAS. Furthermore, antibody cross-reactivity to cardiac myosin was comparable in both GGS- and GAS-exposed animals, providing additional evidence that GGS can induce and/or exacerbate ARF/RHD.

A systematic and functional classification of Streptococcus pyogenes that serves as a new tool for molecular typing and vaccine development.

Streptococcus pyogenes ranks among the main causes of mortality from bacterial infections worldwide. Currently there is no vaccine to prevent diseases such as rheumatic heart disease and invasive streptococcal infection. The streptococcal M protein that is used as the substrate for epidemiological typing is both a virulence factor and a vaccine antigen. Over 220 variants of this protein have been described, making comparisons between proteins difficult, and hindering M protein-based vaccine development. A functional classification based on 48 emm-clusters containing closely related M proteins that share binding and structural properties is proposed. The need for a paradigm shift from type-specific immunity against S. pyogenes to emm-cluster based immunity for this bacterium should be further investigated. Implementation of this emm-cluster-based system as a standard typing scheme for S. pyogenes will facilitate the design of future studies of M protein function, streptococcal virulence, epidemiological surveillance, and vaccine development.

DrsG from Streptococcus dysgalactiae subsp. equisimilis inhibits the antimicrobial peptide LL-37.

SIC and DRS are related proteins present in only 4 of the >200 Streptococcus pyogenes emm types. These proteins inhibit complement-mediated lysis and/or the activity of certain antimicrobial peptides (AMPs). A gene encoding a homologue of these proteins, herein called DrsG, has been identified in the related bacterium Streptococcus dysgalactiae subsp. equisimilis. Here we show that geographically dispersed isolates representing 14 of 50 emm types examined possess variants of drsG. However, not all isolates within the drsG-positive emm types possess the gene. Sequence comparisons also revealed a high degree of conservation in different S. dysgalactiae subsp. equisimilis emm types. To examine the biological activity of DrsG, recombinant versions of two major DrsG variants, DrsGS and DrsGL, were expressed and purified. Western blot analysis using antisera raised to these proteins demonstrated both variants to be expressed and secreted into culture supernatants. Unlike SIC, but similar to DRS, DrsG does not inhibit complement-mediated lysis. However, like both SIC and DRS, DrsG is a ligand of the cathelicidin LL-37 and is inhibitory to its bactericidal activity in in vitro assays. Conservation of prolines in the C-terminal region also suggests that these residues are important in the biology of this family of proteins. This is the first report demonstrating the activity of an AMP-inhibitory protein in S. dysgalactiae subsp. equisimilis and suggests that inhibition of AMP activity is the primary function of this family of proteins. The acquisition of the complement-inhibitory activity of SIC may reflect its continuing evolution.

Differences in virulence repertoire and cell invasive potential of group A Streptococcus emm1-2 in comparison to emm1 genotype.

Group A streptococcus (GAS, Streptococcus pyogenes) type emm1 is widely associated with streptococcal invasive disease. This type is prevalent worldwide but is rare in India. Instead, emm1-2 type which is closely related to emm1 but is a distinct type is more prevalent. Although emm1 has been well characterized, information available on emm1-2 is rare. In this study we present a comparative study of both types. DNA microarray analysis showed segregation of emm1 and emm1-2 isolates into two distinct clusters. Out of 229 arrayed genes, 83-87% were present, 6-9% absent and 4-8% genes were ambiguous in emm1 isolates. emm1-2 strains harboured only 68-77%, 11-13% were absent and 10-20% ambiguous genes. Fourteen genes, present in all emm1, were completely absent in the emm1-2 isolates. sfb1 is a gene which encodes for Streptococcal fibronectin binding adhesin and invasin which has restricted distribution among different emm types of GAS. A variant of sfb1 (sfb1-2) was the only gene which was present in all emm1-2 isolates, but absent from all emm1 strains. Sfb1 and Sfb1-2 differ in sequences in the aromatic domain and the proline rich repeat region, whereas the fibronectin binding region was conserved and exhibited similar fibronectin binding activity. The presence of Sfb1-2 in emm1-2 strains was concomitant with significantly higher fibronectin-binding and invasion efficiency of HEp-2 cells when compared to emm1 isolates. The role of Sfb1-2 in invasion was confirmed by latex bead assay. emm1-2 isolates follow membrane ruffling mechanism during invasion and intracellularly follow classical endocytic pathway. Further studies are required to understand the correlation between the presence of emm1-2 isolates and the disease pattern in North India.

Molecular markers for the study of streptococcal epidemiology.

Diseases caused by Streptococcus pyogenes (Group A streptococcus, GAS) range from superficial infections such as pharyngitis and impetigo to potentially fatal rheumatic heart disease and invasive disease. Studies spanning emm-typing surveillance to population genomics are providing new insights into the epidemiology, pathogenesis, and biology of this organism. Such studies have demonstrated the differences that exist in the epidemiology of streptococcal disease between developing and developed nations. In developing nations, where streptococcal disease is endemic, the diversity of GAS emm-types circulating is much greater than that found in developed nations. An association between emm-type and disease, as observed in developed countries is also lacking. Intriguingly, comparative genetic studies suggest that emm-type is not always a good predictor of the evolutionary relatedness of geographically distant isolates. A view of GAS as a highly dynamic organism, in possession of a core set of virulence genes that contribute to host niche specialization and common pathogenic processes, augmented by accessory genes that change the relative virulence of specific lineages is emerging. Our inability to definitively identify genetic factors that contribute to specific disease outcome underscores the complex nature of streptococcal diseases.

Mechanisms that potentially contribute to the development of post-streptococcal glomerulonephritis.

Post-streptococcal glomerulonephritis (PSGN) is primarily associated with preceding Group A streptococcal skin or throat infections, now mainly observed in economically disadvantaged communities. This condition significantly predisposes individuals to later-life chronic kidney disease and concurrent renal complications, with the elderly experiencing increased severity and less favourable outcomes. Streptococcal pyrogenic exotoxin B and nephritis associated plasmin receptor are identified nephritogenic antigens (nephritogens). Pathogenesis of PSGN is multifactorial. It can involve the formation of antigen-antibody immune complexes, causing inflammatory damage to renal glomeruli. Deposition of circulating immune complexes or in situ formation of immune complexes in glomeruli, or both, results in glomerulonephritis. Additionally, molecular mimicry is hypothesised as a mechanism, wherein cross-reactivity between anti-streptococcal antibodies and glomerular intrinsic matrix proteins leads to glomerulonephritis. Besides, as observed in clinical studies, streptococcal inhibitor of complement, a streptococcal-secreted protein, can also be associated with PSGN. However, the interplay between these streptococcal antigens in the pathogenesis of PSGN necessitates further investigation. Despite the clinical significance of PSGN, the lack of credible animal models poses challenges in understanding the association between streptococcal antigens and the disease process. This review outlines the postulated mechanisms implicated in the development of PSGN with possible therapeutic approaches.

Inter-species gene flow drives ongoing evolution of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

Development and pre-clinical evaluation of a Zika virus diagnostic for low resource settings.

Introduction: Zika virus (ZIKV) is a re-emerging flavivirus that poses a significant public health threat. ZIKV exhibits a wide array of non-vector borne human transmission routes, such as sexual transmission, transplacental transmission and blood transfusion. Detection and surveillance of ZIKV is considered paramount in prevention of major outbreaks. With the majority of cases reported in low-resource locations, simple, low-cost detection methods are considered highly desirable. Materials and Methods: Here we have developed a sensitive and specific ZIKV diagnostic using reverse transcription recombinase-aided amplification (RT-RAA) coupled with lateral flow detection (LFD) targeting a highly conserved region of the ZIKV NS1 gene. Results: We show our rapid, isothermal-ZIKV-diagnostic (Iso-ZIKV-Dx) can detect 500 copies of synthetic ZIKV RNA/µL in under 30 min at a constant 39°C. Using simulated urine samples, we observed that Iso-ZIKV-Dx also detects as low as 34.28 RNA copies/reaction of ZIKV (MR766 strain). Specificity testing confirmed that our test does not detect any co-circulating flaviviruses (dengue, West Nile, Japanese encephalitis, Murray Valley encephalitis and yellow fever viruses) or chikungunya virus. Sample processing results show complete inactivation of ZIKV (MR766 strain) in 5 min at room temperature using our novel viral RNA sample preparation reagent. Furthermore, lateral flow strips testing demonstrates positive diagnoses in as little as 5 min in running buffer. Discussion: Contrary to conventional RT-qPCR, our Iso-ZIKV-Dx does not require expensive machinery, specialised laboratory settings or extensively trained personnel. Pre-clinical evaluation demonstrates that our test exhibits robust, in-field capabilities without compromising sensitivity or specificity. When compared to the gold-standard RT-qPCR, our Iso-ZIKV-Dx test offers an array of applications that extend beyond diagnostics alone, including potential for surveillance and monitoring of ZIKV vector competency.

In Search of the Holy Grail: A Specific Diagnostic Test for Rheumatic Fever.

Current diagnosis of Acute Rheumatic Fever and Rheumatic Heart Disease (ARF/RHD) relies on a battery of clinical observations aided by technologically advanced diagnostic tools and non-specific laboratory tests. The laboratory-based assays fall into two categories: those that (1) detect "evidence of preceding streptococcal infections" (ASOT, anti-DNAse B, isolation of the Group A Streptococcus from a throat swab) and (2) those that detect an ongoing inflammatory process (ESR and CRP). These laboratory tests are positive during any streptococcal infection and are non-specific for the diagnosis of ARF/RHD. Over the last few decades, we have accumulated considerable knowledge about streptococcal biology and the immunopathological mechanisms that contribute to the development, progression and exacerbation of ARF/RHD. Although our knowledge is incomplete and many more years will be devoted to understanding the exact molecular and cellular mechanisms involved in the spectrum of clinical manifestations of ARF/RHD, in this commentary we contend that there is sufficient understanding of the disease process that using currently available technologies it is possible to identify pathogen associated peptides and develop a specific test for ARF/RHD. It is our view that with collaboration and sharing of well-characterised serial blood samples from patients with ARF/RHD from different regions, antibody array technology and/or T-cell tetramers could be used to identify streptococcal peptides specific to ARF/RHD. The availability of an appropriate animal model for this uniquely human disease can further facilitate the determination as to whether these peptides are pathognomonic. Identification of such peptides will also facilitate testing of potential anti-streptococcal vaccines for safety and avoid potential candidates that may pre-dispose potential vaccine recipients to adverse outcomes. Such peptides can also be readily incorporated into a universally affordable point of care device for both primary and tertiary care.

Requirements for a Robust Animal Model to Investigate the Disease Mechanism of Autoimmune Complications Associated With ARF/RHD.

The pathogenesis of Acute Rheumatic Fever/Rheumatic Heart Disease (ARF/RHD) and associated neurobehavioral complications including Sydenham's chorea (SC) is complex. Disease complications triggered by Group A streptococcal (GAS) infection are confined to human and determining the early events leading to pathology requires a robust animal model that reflects the hallmark features of the disease. However, modeling these conditions in a laboratory animal, of a uniquely human disease is challenging. Animal models including cattle, sheep, pig, dog, cat, guinea pigs rats and mice have been used extensively to dissect molecular mechanisms of the autoimmune inflammatory responses in ARF/RHD. Despite the characteristic limitations of some animal models, several rodent models have significantly contributed to better understanding of the fundamental mechanisms underpinning features of ARF/RHD. In the Lewis rat autoimmune valvulitis model the development of myocarditis and valvulitis with the infiltration of mononuclear cells along with generation of antibodies that cross-react with cardiac tissue proteins following exposure to GAS antigens were found to be similar to ARF/RHD. We have recently shown that Lewis rats injected with recombinant GAS antigens simultaneously developed cardiac and neurobehavioral changes. Since ARF/RHD is multifactorial in origin, an animal model which exhibit the characteristics of several of the cardinal diagnostic criteria observed in ARF/RHD, would be advantageous to determine the early immune responses to facilitate biomarker discovery as well as provide a suitable model to evaluate treatment options, safety and efficacy of vaccine candidates. This review focuses on some of the common small animals and their advantages and limitations.

Group A streptococcal antigen exposed rat model to investigate neurobehavioral and cardiac complications associated with post-streptococcal autoimmune sequelae.

Background: The neuropsychiatric disorders due to post-streptococcal autoimmune complications such as Sydenham's chorea (SC) are associated with acute rheumatic fever and rheumatic heart disease (ARF/RHD). An animal model that exhibits characteristics of both cardiac and neurobehavioral defects in ARF/RHD would be an important adjunct for future studies. Since age, gender, strain differences, and genotypes impact on the development of autoimmunity, we investigated the behavior of male and female Wistar and Lewis rat strains in two age cohorts (<6 weeks and >12 weeks) under normal husbandry conditions and following exposure to group A streptococcus (GAS). Methods: Standard behavioral assessments were performed to determine the impairments in fine motor control (food manipulation test), gait and balance (beam walking test), and obsessive-compulsive behavior (grooming and marble burying tests). Furthermore, electrocardiography, histology, and behavioral assessments were performed on male and female Lewis rats injected with GAS antigens. Results: For control Lewis rats there were no significant age and gender dependent differences in marble burying, food manipulation, beam walking and grooming behaviors. In contrast significant age-dependent differences were observed in Wistar rats in all the behavioral tests except for food manipulation. Therefore, Lewis rats were selected for further experiments to determine the effect of GAS. After exposure to GAS, Lewis rats demonstrated neurobehavioral abnormalities and cardiac pathology akin to SC and ARF/RHD, respectively. Conclusion: We have characterised a new model that provides longitudinal stability of age-dependent behavior, to simultaneously investigate both neurobehavioral and cardiac abnormalities associated with post-streptococcal complications.

In silico characterisation of stand-alone response regulators of Streptococcus pyogenes.

Bacterial "stand-alone" response regulators (RRs) are pivotal to the control of gene transcription in response to changing cytosolic and extracellular microenvironments during infection. The genome of group A Streptococcus (GAS) encodes more than 30 stand-alone RRs that orchestrate the expression of virulence factors involved in infecting multiple tissues, so causing an array of potentially lethal human diseases. Here, we analysed the molecular epidemiology and biological associations in the coding sequences (CDSs) and upstream intergenic regions (IGRs) of 35 stand-alone RRs from a collection of global GAS genomes. Of the 944 genomes analysed, 97% encoded 32 or more of the 35 tested RRs. The length of RR CDSs ranged from 297 to 1587 nucleotides with an average nucleotide diversity (π) of 0.012, while the IGRs ranged from 51 to 666 nucleotides with average π of 0.017. We present new evidence of recombination in multiple RRs including mga, leading to mga-2 switching, emm-switching and emm-like gene chimerization, and the first instance of an isolate that encodes both mga-1 and mga-2. Recombination was also evident in rofA/nra and msmR loci with 15 emm-types represented in multiple FCT (fibronectin-binding, collagen-binding, T-antigen)-types, including novel emm-type/FCT-type pairings. Strong associations were observed between concatenated RR allele types, and emm-type, MLST-type, core genome phylogroup, and country of sampling. No strong associations were observed between individual loci and disease outcome. We propose that 11 RRs may form part of future refinement of GAS typing systems that reflect core genome evolutionary associations. This subgenomic analysis revealed allelic traits that were informative to the biological function, GAS strain definition, and regional outbreak detection.

Potent Antibacterial Prenylated Acetophenones from the Australian Endemic Plant Acronychia crassipetala.

Acronychia crassipetala is an endemic plant species in Australia. Its phytochemistry and therapeutic properties are underexplored. The hexane extract of the fruit A. crassipetala T. G. Hartley was found to inhibit the growth of the Gram-positive bacteria Staphylococcus aureus. Following bio-activity guided fractionation, two prenylated acetophenones, crassipetalonol A (1) and crassipetalone A (2), were isolated. Their structures were determined mainly by NMR and MS spectroscopic analyses. This is the first record of the isolation and structural characterisation of secondary metabolites from the species A. crassipetala. Their antibacterial and cytotoxic assessments indicated that the known compound (2) had more potent antibacterial activity than the antibiotic chloramphenicol, while the new compound (1) showed moderate cytotoxicity.

A novel integrative conjugative element mediates genetic transfer from group G Streptococcus to other {beta}-hemolytic Streptococci.

Lateral gene transfer is a significant contributor to the ongoing evolution of many bacterial pathogens, including beta-hemolytic streptococci. Here we provide the first characterization of a novel integrative conjugative element (ICE), ICESde3396, from Streptococcus dysgalactiae subsp. equisimilis (group G streptococcus [GGS]), a bacterium commonly found in the throat and skin of humans. ICESde3396 is 64 kb in size and encodes 66 putative open reading frames. ICESde3396 shares 38 open reading frames with a putative ICE from Streptococcus agalactiae (group B streptococcus [GBS]), ICESa2603. In addition to genes involves in conjugal processes, ICESde3396 also carries genes predicted to be involved in virulence and resistance to various metals. A major feature of ICESde3396 differentiating it from ICESa2603 is the presence of an 18-kb internal recombinogenic region containing four unique gene clusters, which appear to have been acquired from streptococcal and nonstreptococcal bacterial species. The four clusters include two cadmium resistance operons, an arsenic resistance operon, and genes with orthologues in a group A streptococcus (GAS) prophage. Streptococci that naturally harbor ICESde3396 have increased resistance to cadmium and arsenate, indicating the functionality of genes present in the 18-kb recombinogenic region. By marking ICESde3396 with a kanamycin resistance gene, we demonstrate that the ICE is transferable to other GGS isolates as well as GBS and GAS. To investigate the presence of the ICE in clinical streptococcal isolates, we screened 69 isolates (30 GGS, 19 GBS, and 20 GAS isolates) for the presence of three separate regions of ICESde3396. Eleven isolates possessed all three regions, suggesting they harbored ICESde3396-like elements. Another four isolates possessed ICESa2603-like elements. We propose that ICESde3396 is a mobile genetic element that is capable of acquiring DNA from multiple bacterial sources and is a vehicle for dissemination of this DNA through the wider beta-hemolytic streptococcal population.

Skin colonization at peripheral intravenous catheter insertion sites increases the risk of catheter colonization and infection.

BACKGROUND: Peripheral intravenous catheters (PIVCs) break the skin barrier, and preinsertion antiseptic disinfection and sterile dressings are used to reduce risk of catheter-related bloodstream infection (CRBSI). In this study, the impact of PIVC skin site colonization on tip colonization and the development of CRBSI was investigated. METHODS: A total of 137 patients' PIVC skin site swabs and paired PIVC tips were collected at catheter removal, cultured, and bacterial species and clonality were identified. RESULTS: Of 137 patients, 45 (33%) had colonized skin sites and/or PIVC tips. Of 16 patients with paired colonization of both the skin site and PIVC tips, 11 (69%) were colonized with the same bacterial species. Of these, 77% were clonally related, including 1 identical clone of Pseudomonas aeruginosa in a patient with systemic infection and the same organism identified in blood culture. CONCLUSIONS: The results demonstrate that opportunistic pathogen colonization at the skin site poses a significant risk for PIVC colonization and CRBSI. Further research is needed to improve current preinsertion antiseptic disinfection of PIVC skin site and the sterile insertion procedure to potentially reduce PIVC colonization and infection risk.

In silico characterisation of the two-component system regulators of Streptococcus pyogenes.

Bacteria respond to environmental changes through the co-ordinated regulation of gene expression, often mediated by two-component regulatory systems (TCS). Group A Streptococcus (GAS), a bacterium which infects multiple human body sites and causes multiple diseases, possesses up to 14 TCS. In this study we examined genetic variation in the coding sequences and non-coding DNA upstream of these TCS as a method for evaluating relationships between different GAS emm-types, and potential associations with GAS disease. Twelve of the 14 TCS were present in 90% of the genomes examined. The length of the intergenic regions (IGRs) upstream of TCS coding regions varied from 39 to 345 nucleotides, with an average nucleotide diversity of 0.0064. Overall, IGR allelic variation was generally conserved with an emm-type. Subsequent phylogenetic analysis of concatenated sequences based on all TCS IGR sequences grouped genomes of the same emm-type together. However grouping with emm-pattern and emm-cluster-types was much weaker, suggesting epidemiological and functional properties associated with the latter are not due to evolutionary relatedness of emm-types. All emm5, emm6 and most of the emm18 genomes, all historically considered rheumatogenic emm-types clustered together, suggesting a shared evolutionary history. However emm1, emm3 and several emm18 genomes did not cluster within this group. These latter emm18 isolates were epidemiologically distinct from other emm18 genomes in study, providing evidence for local variation. emm-types associated with invasive disease or nephritogenicity also did not cluster together. Considering the TCS coding sequences (cds), correlation with emm-type was weaker than for the IGRs, and no strong correlation with disease was observed. Deletion of the malate transporter, maeP, was identified that serves as a putative marker for the emm89.0 subtype, which has been implicated in invasive outbreaks. A recombination-related, subclade-forming DNA motif was identified in the putative receiver domain of the Spy1556 response regulator that correlated with throat-associated emm-pattern-type A-C strains.