Report of fourth project year (2016) - Summary

The overall goal of INFECT is to advance our understanding of the pathophysiological mechanisms, prognosis, and diagnosis of the multifactorial highly lethal necrotizing soft tissue infections (NSTIs). NSTI’s are rapidly spreading infections that may cause extensive soft tissue or limb loss, multiorgan failure and are associated with a considerable fatality rate. It is undisputed that rapid diagnosis and prompt intervention is directly related to survival. The initial presentation may be limited to unspecific symptoms such as tenderness, swelling, erythema and pain. Thus, diagnosis and management are difficult due to heterogeneity in clinical presentation, in co-morbidities and in microbiological aetiology. There is an urgent need for novel diagnostic and therapeutic strategies in order to improve outcome of NSTIs. To achieve this, a comprehensive and integrated knowledge of diagnostic features, causative microbial agent, treatment strategies, and pathogenic mechanisms (host and bacterial disease traits and their underlying interaction network) is required. INFECT is designed to obtain such insights through an integrated systems biology approach in patients and different clinically relevant experimental models.

Specific objectives of INFECT are to:

  • Unravel specific mechanisms underlying diseases responses through a bottom-up systems approach applied to clinically relevant experimental models
  • Apply a top-down systems biology approach to NSTI patient samples to pin-point key host and pathogen factors involved in the onset and development of infection
  • Identify and quantify disease signatures and underlying networks that contribute to disease outcome
  • Exploit identified disease traits for the innovation of optimized diagnostic tools
  • Translate the advanced knowledge generated into evidence-based guidelines for classification and management, and novel therapeutic strategies

Summary of the project for the fourth project year (2016)

During this reporting period, the main objective of WP1 was to explore novel therapeutic interventions targeting identified key regulatory networks contributing to pathogenesis in the NSTI murine models. Therefore, partner 15 performed transcriptome analysis of high throughput RNA sequencing data of skin samples from mice infected with clinical NSTI/STSS strains of Group A Streptococcus (GAS), which revealed significant down regulation of the fatty acid and nuclear hormone receptor, Peroxisome Proliferator Activated Receptor γ (PPARγ), while transcripts of the key proinflammatory mediators IL-1b and IFN-g were significantly upregulated during NSTI. Parsing the differentially expressed genes using Ingenuity Pathway Analysis tools revealed that both IL-1b and PPARg were key upstream regulators driving the differential responses, and, therefore, potential candidates for intervention to combat susceptibility and severe outcomes of NSTI. PPARg is activated by natural ligands such as fatty acids, eicosanoids, oxidized fatty acids, and by pharmacological compounds such as thiazolidinediones. The unexpected linkage between downregulation of PPARg and susceptibility to severe outcomes of NSTI was of particular interest and physiologically relevant that might have significant implications in determining novel therapeutic strategies. Based on the hypothesis that GAS infections somehow limit the availiability of PPARg ligands and thereby suppress PPARg expression, interventions designed to enhance PPARg expression is a possible novel strategy to attenuate severe outcomes of NSTI. Specifically, pharmacological intervention therapy using thiazolidinediones (PPARg agonists) used to treat and ameliorate Type 2 Diabetes or other synthetic PPARg ligands are likely to provide better benefits and favourable outcomes. In this reporting period, in addition to evaluating the effect of the routinely administered IvIg as intervention, partner 15 also investigated the beneficial effects of PPARg ligands (agonists), Farnesol (FN) and Pioglitazone (PZ) as novel therapeutic interventions and monitored the severity of lesions and survival during NSTI. The effect of the routinely administered IvIg alone or IvIg + FN in addition to administration of PZ, ciprofloxazin (Cipro), or Cipro + FN as possible means to drive protective responses thereby attenuating severity and promoting survival during NSTI was investigated by partner 15 during this reporting period.

In WP2 NSTI patient enrolment and biobank sample collection is achieved. All 5 clinical centres (partners 2-6) have contributed and the total patient enrolment amounts 383 patients. Thus, we estimate that our goal target of 400 patients will be reached during spring of 2017. Two sets of “clean” data sets from the WP2 clinical database (i.e. 200 patient records) have now been exported from the WP2 group to partners 9, 10, 11 and 14 for subsequent analysis. Statistical analysis plans for the different scientific reports – based on primarily clinical data sets – have now been developed and pre-tested using the 200 patient records; thus, providing the foundation for the future for future evidence-based guidelines for the best management of NSTI infections. WP2 partners have interacted closely with the experimental (partners 1, 8, 10, 15), modelling partners (9, 11, 14) and SME partner (partner 16) to support their respective WPs and ensure that all clinical aspects are taken into account; thus allowing for full exploitation of the data and samples.

The main objective of WP3 is to identify specific pathogen and host disease signatures. On the pathogen side one specific objective is the application of various standard typing methods to establish a database containing virulence specific information of isolated bacterial pathogens involved in NSTI. During the reporting period, the dominant role of the species Streptococcus pyogenes, specifically of serotype M1 as main causative of severe NSTIs, as well as the underestimated pathogenic potential of Streptococcus dysgalactiae ssp. equisimilis within the Scandinavian NSTI cohort could be further confirmed. The frequent occurrence of multi-species NSTI cases in the INFECT cohort was specifically addressed by partner 8 using a comprehensive amplicon based species composition analysis, which was not only able to clearly identify NSTI cases associated with Staphylococcus aureus as main pathogen, but also provided new insights into the community composition of mixed-species tissue infections, which enabled a recently started pathogen specific transcriptome analysis with special focus on the comparison of mono and multi-species infections. A comprehensive whole genome comparison including 65 sequenced streptococcal and staphylococcal NSTI isolates was performed, targeting to identify bacterial genes essential for the development of an NSTI (partners 8 and 10). The recently identified role of streptococcal biofilm formation during NSTI development (partner 1) was further investigated by partner 8 through an examination of the regulatory processes behind biofilm development and maturation with special focus on streptococcal exotoxins as bacterial signal molecules. This approach identified a novel function of SpeA as inducer of controlled biofilm disruption and bacterial spreading as potential new target for the specific treatment of S. pyogenes tissue biofilms. Partner 1 explored the role of host neutrophils by an examination of degranulation responses to specific stimuli induced by the dominant NSTI pathogen S. pyogenes. This approach identified phosphoglycerate kinase as novel potent inducer of neutrophil degranulation with special dependency of the bacterial SpeB protease activity. A transcriptomic examination of the efficacy and the impact of an applied HBO treatment on the cellular level directly at the side of infection showed the bactericidal effects of this treatment and identified hydrogen peroxide and hydroxyl radicals as the major harmful factors, which indicates that an applied HBO treatment can contribute to the bacterial killing in infected tissue and constitutes a general benefit for the disease outcome (partner 8).

During this reporting period, the main objectives of WP5 were to follow-up on results obtained by partner 15 in WP1, namely identification of IL1b as a key pathway involved in severity of NSTI, as well as to gain insight into pathogenic mechanisms focusing on macrophage characteristics at the local tissue site. Work achieved by partner 1 during the reporting period has included analyses of tissue biopsies from NSTI patients for bacterial and host factors. The analyses provided novel insight including identification of biofilm in S. pyogenes infected NSTI, in particular in biopsies characterized by high bacterial load and high inflammation. Work together with partner 14 is focusing on the simulation of biofilm in tissue to generate a model of metabolic requirements/triggers. It furthermore provided clinical support for the relevance of the finding of the IL1b pathway determining severity of NSTI in the murine experimental model (WP1), inasmuch as IL1b was highly expressed in patient tissue. Partner 1 also have developed novel techniques to allow for characterization of macrophage phenotypes at the single cell, and the data indicate a skewing in the macrophage phenotype as a consequence of infection. These studies are onoing and further validated by further studies of patient samples (WP3 and WP5) as well as in our experimental models (WP1 and WP6).

The overall aim of WP6 is to establish a clinically relevant, robust and reproducible multicellular system, a so called organotypic model, resembling normal human skin tissue that can be used for experimental infections and test of therapeutic strategies (partner 1). During the 1st, 2nd and 3rd period, partner 1 focused the efforts on establishing and using the skin oragnotypic model to identify bacterial and host determinants of pathogenesis in NSTI with pathogenic strains of Streptococcus pyogenes, Streptococcus dysgalactiae subspecies equisimilis (SDSE), and Staphylococcus aureus s (partner 1-6 and 10). Modelling infections with S. aureus revealed how a single amino acid substitution in the histidine kinase receptor AgrC of ST22 strains determines virulence properties and infection outcome (partner 1). This was further validated in the in vivo NSTI murine model (partner 15). Further, studies investigating what host-derived components are required for S. pyogenes biofilm formation in human skin was initiated during the 4th period (partner 1 and 14). Also, skin tissue models infected with S. pyogenes were used to confirm data from the experimental murine model showing IL1b as a key network involved in modulating the differential susceptibility to GAS NSTIs (partners 1 and 15). Tissue models were further developed and established to contain also monocyte-derived cells. Such models were used in experiments with the INFECT 2006 isolate (S. pyogenes) to generate host transcriptional profiles (partner 1) that will be integrated in the data driven computational modelling platform (partners 9, 11, 14). Over all, the progress of work is well in line with the objectives and tasks of this work package and the work towards identifying pathways contributing to histopathology chages in tissue. Results from the experiments performed were compiled into three separate publications.

WP7 focuses on developing a novel diagnostics approach in NSTI by applying compact sequencing (pathogen detection - DNA) and compact profiling (disease traits – antigens) – both multiplex diagnostic technologies. Partner 16 has extended and verified the Pathogen ID test as well as the Inflammation and Organ Monitoring tests. Beside that, a prototype for screening of antibodies against 18 different exotoxins of Streptococcus pyogenes had been developed by partner 16 together with partner 8 to stratify patients especially vulnerable for this kind of infection. The clinical utility of that test has still to be decided by the clinical partners. Further improvements have been performed including the device (hyborg) running the tests. It was also realized, that for the specific situation of diagnosing and treating NSTI, a DNA based identification of the causing pathogen is still too cumbersome to be implemented in clinical routine. Therefore it was agreed upon to focus on the stratification / monitoring of patients based on Cube Dx’ inflammation (and organ) monitoring and eventually on the screening of anti-exotoxins antibodies. For the inflammation and organ monitoring panel, partner 16 has extended the 9 biomarkers to include 13 biomarkers (selected based on the criteria clinical utility, technical feasibility, reproducibility of prototype production).

The ultimate objectives of WP8 is to ensure an efficient dissemination and exploitation of knowledge generated within the INFECT project. During this reporting period, a broad range of different contributions to dissemination activities, including among others presentations at scientific meeting, patient organisation activities, lecturing and publications have been undertaken. Scientific publications in well-renowned journals have been achieved for all scientific WP and includes the participation of all partners. In addition, the work has been presented at numerous scientific conferences as well as meetings addressing the civil society and/or policy makers. The emphasis has been on disseminating the knowledge generated in the project, increasing the awareness of these life-threatening infections and working towards the implementation of systems medicine in infectious disease/intensive care. At the international Systems Medicine conference in Berlin (October 2016), two presentations from the INFECT consortium were among the top 10% selected for oral presentation; thus demonstrating the high quality of the work produced.

The aim of WP9 is to provide an efficient project management for INFECT and its 14 partners. Main activities during this period focused on ensuring efficient data generation as well as sharing and integration of data between the participants in the project. Beyond this the management included admistrative activities to ensure full compliance with all legal and EC requirements. Also, an ethical review of the work conducted during the forth period has been conducted by the external advisor. During this reporting period, several meetings between clinical and data modelling partners (partners 2-6, 9, 11, 16), as well as meetings between the experimental/modelling partners (partners 1, 8, 9, 11 and 15). It also included regular skype conferences with a focus on data generation, standardization, sharing, integration, and interpretation between experimental partners (partners 1, 8-11, 14 and 15). Overall, the project has generated lots of new and exciting data, which has instigated new collaborations and new hypothesis that are currently being tested in the various WPs.