Dynamic MAIT Cell Recovery after Severe COVID-19 Is Transient with Signs of Heterogeneous Functional Anomalies.

Mucosal-associated invariant T (MAIT) cells are an abundant population of unconventional T cells in humans and play important roles in immune defense against microbial infections. Severe COVID-19 is associated with strong activation of MAIT cells and loss of these cells from circulation. In the present study, we investigated the capacity of MAIT cells to recover after severe COVID-19. In longitudinal paired analysis, MAIT cells initially rebounded numerically and phenotypically in most patients at 4 mo postrelease from the hospital. However, the rebounding MAIT cells displayed signs of persistent activation with elevated expression of CD69, CD38, and HLA-DR. Although MAIT cell function was restored in many patients, a subgroup displayed a predominantly PD-1high functionally impaired MAIT cell pool. This profile was associated with poor expression of IFN-γ and granzyme B in response to IL-12 + L-18 and low levels of polyfunctionality. Unexpectedly, although the overall T cell counts recovered, normalization of the MAIT cell pool failed at 9-mo follow-up, with a clear decline in MAIT cell numbers and a further increase in PD-1 levels. Together, these results indicate an initial transient period of inconsistent recovery of MAIT cells that is not sustained and eventually fails. Persisting MAIT cell impairment in previously hospitalized patients with COVID-19 may have consequences for antimicrobial immunity and inflammation and could potentially contribute to post-COVID-19 health problems.

Reduced interleukin-18 secretion by human monocytic cells in response to infections with hyper-virulent Streptococcus pyogenes.

BACKGROUND: Streptococcus pyogenes (group A streptococcus, GAS) causes a variety of diseases ranging from mild superficial infections of the throat and skin to severe invasive infections, such as necrotizing soft tissue infections (NSTIs). Tissue passage of GAS often results in mutations within the genes encoding for control of virulence (Cov)R/S two component system leading to a hyper-virulent phenotype. Dendritic cells (DCs) are innate immune sentinels specialized in antigen uptake and subsequent T cell priming. This study aimed to analyze cytokine release by DCs and other cells of monocytic origin in response to wild-type and natural covR/S mutant infections. METHODS: Human primary monocyte-derived (mo)DCs were used. DC maturation and release of pro-inflammatory cytokines in response to infections with wild-type and covR/S mutants were assessed via flow cytometry. Global proteome changes were assessed via mass spectrometry. As a proof-of-principle, cytokine release by human primary monocytes and macrophages was determined. RESULTS: In vitro infections of moDCs and other monocytic cells with natural GAS covR/S mutants resulted in reduced secretion of IL-8 and IL-18 as compared to wild-type infections. In contrast, moDC maturation remained unaffected. Inhibition of caspase-8 restored secretion of both molecules. Knock-out of streptolysin O in GAS strain with unaffected CovR/S even further elevated the IL-18 secretion by moDCs. Of 67 fully sequenced NSTI GAS isolates, 28 harbored mutations resulting in dysfunctional CovR/S. However, analyses of plasma IL-8 and IL-18 levels did not correlate with presence or absence of such mutations. CONCLUSIONS: Our data demonstrate that strains, which harbor covR/S mutations, interfere with IL-18 and IL-8 responses in monocytic cells by utilizing the caspase-8 axis. Future experiments aim to identify the underlying mechanism and consequences for NSTI patients.

Mucosa-Associated Invariant T Cell Hypersensitivity to Staphylococcus aureus Leukocidin ED and Its Modulation by Activation.

Mucosa-associated invariant T (MAIT) cells recognize bacterial riboflavin metabolite Ags presented by MHC class Ib-related protein (MR1) and play important roles in immune control of microbes that synthesize riboflavin. This includes the pathobiont Staphylococcus aureus, which can also express a range of virulence factors, including the secreted toxin leukocidin ED (LukED). In this study, we found that human MAIT cells are hypersensitive to LukED-mediated lysis and lost on exposure to the toxin, leaving a T cell population devoid of MAIT cells. The cytolytic effect of LukED on MAIT cells was rapid and occurred at toxin concentrations lower than those required for toxicity against conventional T cells. Furthermore, this coincided with high MAIT cell expression of CCR5, and loss of these cells was efficiently inhibited by the CCR5 inhibitor maraviroc. Interestingly, exposure and preactivation of MAIT cells with IL-12 and IL-18, or activation via TCR triggering, partially protected from LukED toxicity. Furthermore, analysis of NK cells indicated that LukED targeted the mature cytotoxic CD57+ NK cell subset in a CCR5-independent manner. Overall, these results indicate that LukED efficiently eliminates immune cells that can respond rapidly to S. aureus in an innate fashion without the need for clonal expansion, and that MAIT cells are exceptionally vulnerable to this toxin. Thus, the findings support a model where LukED secretion may allow S. aureus to avoid recognition by the rapid cell-mediated responses mediated by MAIT cells and NK cells.

Major alterations in the mononuclear phagocyte landscape associated with COVID-19 severity.

Dendritic cells (DCs) and monocytes are crucial mediators of innate and adaptive immune responses during viral infection, but misdirected responses by these cells may contribute to immunopathology. Here, we performed high-dimensional flow cytometry-analysis focusing on mononuclear phagocyte (MNP) lineages in SARS-CoV-2-infected patients with moderate and severe COVID-19. We provide a deep and comprehensive map of the MNP landscape in COVID-19. A redistribution of monocyte subsets toward intermediate monocytes and a general decrease in circulating DCs was observed in response to infection. Severe disease coincided with the appearance of monocytic myeloid-derived suppressor cell-like cells and a higher frequency of pre-DC2. Furthermore, phenotypic alterations in MNPs, and their late precursors, were cell-lineage-specific and associated either with the general response against SARS-CoV-2 or COVID-19 severity. This included an interferon-imprint in DC1s observed in all patients and a decreased expression of the coinhibitory molecule CD200R in pre-DCs, DC2s, and DC3 subsets of severely sick patients. Finally, unsupervised analysis revealed that the MNP profile, alone, pointed to a cluster of COVID-19 nonsurvivors. This study provides a reference for the MNP response to SARS-CoV-2 infection and unravels mononuclear phagocyte dysregulations associated with severe COVID-19.

High-dimensional profiling reveals phenotypic heterogeneity and disease-specific alterations of granulocytes in COVID-19.

Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate immune responses play an important role in the disease development. A dysregulated inflammatory state has been proposed as a key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients is lacking. In this study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in peripheral blood collected from COVID-19 patients during acute and convalescent phases. Severe COVID-19 was associated with increased levels of both mature and immature neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were evident in COVID-19 patients, with altered expression of several receptors involved in activation, adhesion, and migration of granulocytes (e.g., CD62L, CD11a/b, CD69, CD63, CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic signature in the convalescent phase. The identified granulocyte immunotypes correlated with distinct sets of soluble inflammatory markers, supporting pathophysiologic relevance. Furthermore, clinical features, including multiorgan dysfunction and respiratory function, could be predicted using combined laboratory measurements and immunophenotyping. This study provides a comprehensive granulocyte characterization in COVID-19 and reveals specific immunotypes with potential predictive value for key clinical features associated with COVID-19.

Necrotising soft tissue infections. / Nekrotiserende bløtdelsinfeksjoner.

Necrotising soft tissue infections can affect the skin, subcutaneous tissue, superficial fascia, deep fascia and musculature. The infections are severe, they spread quickly and can result in extensive tissue loss. Although rare, morbidity and mortality rates are high. Early clinical identification is crucial for the outcome, and rapid infection control through surgery and targeted antibiotic treatment is needed to save lives. Few prospective clinical trials have been conducted for the treatment of this type of infection. Specific challenges include rapid identification of the condition and the uncertain efficacy of the various treatment options. In this clinical review article, we describe clinical characteristics, diagnostics and treatment.

Systemic immune activation profiles in streptococcal necrotizing soft tissue infections: A prospective multicenter study.

OBJECTIVE: Early stages with streptococcal necrotizing soft tissue infections (NSTIs) are often difficult to discern from cellulitis. Increased insight into inflammatory responses in streptococcal disease may guide correct interventions and discovery of novel diagnostic targets. METHODS: Plasma levels of 37 mediators, leucocytes and CRP from 102 patients with ß-hemolytic streptococcal NSTI derived from a prospective Scandinavian multicentre study were compared to those of 23 cases of streptococcal cellulitis. Hierarchical cluster analyses were also performed. RESULTS: Differences in mediator levels between NSTI and cellulitis cases were revealed, in particular for IL-1ß, TNFα and CXCL8 (AUC >0.90). Across streptococcal NSTI etiologies, eight biomarkers separated cases with septic shock from those without, and four mediators predicted a severe outcome. CONCLUSION: Several inflammatory mediators and wider profiles were identified as potential biomarkers of NSTI. Associations of biomarker levels to type of infection and outcomes may be utilized to improve patient care and outcomes.

COVID-19-specific metabolic imprint yields insights into multiorgan system perturbations.

Corona disease 2019 (COVID-19) affects multiple organ systems. Recent studies have indicated perturbations in the circulating metabolome linked to COVID-19 severity. However, several questions pertain with respect to the metabolome in COVID-19. We performed an in-depth assessment of 1129 unique metabolites in 27 hospitalized COVID-19 patients and integrated results with large-scale proteomic and immunology data to capture multiorgan system perturbations. More than half of the detected metabolic alterations in COVID-19 were driven by patient-specific confounding factors ranging from comorbidities to xenobiotic substances. Systematically adjusting for this, a COVID-19-specific metabolic imprint was defined which, over time, underwent a switch in response to severe acute respiratory syndrome coronavirus-2 seroconversion. Integration of the COVID-19 metabolome with clinical, cellular, molecular, and immunological severity scales further revealed a network of metabolic trajectories aligned with multiple pathways for immune activation, and organ damage including neurological inflammation and damage. Altogether, this resource refines our understanding of the multiorgan system perturbations in severe COVID-19 patients.

Targeted plasma proteomics reveals signatures discriminating COVID-19 from sepsis with pneumonia.

BACKGROUND: COVID-19 remains a major public health challenge, requiring the development of tools to improve diagnosis and inform therapeutic decisions. As dysregulated inflammation and coagulation responses have been implicated in the pathophysiology of COVID-19 and sepsis, we studied their plasma proteome profiles to delineate similarities from specific features. METHODS: We measured 276 plasma proteins involved in Inflammation, organ damage, immune response and coagulation in healthy controls, COVID-19 patients during acute and convalescence phase, and sepsis patients; the latter included (i) community-acquired pneumonia (CAP) caused by Influenza, (ii) bacterial CAP, (iii) non-pneumonia sepsis, and (iv) septic shock patients. RESULTS: We identified a core response to infection consisting of 42 proteins altered in both COVID-19 and sepsis, although higher levels of cytokine storm-associated proteins were evident in sepsis. Furthermore, microbiologic etiology and clinical endotypes were linked to unique signatures. Finally, through machine learning, we identified biomarkers, such as TRIM21, PTN and CASP8, that accurately differentiated COVID-19 from CAP-sepsis with higher accuracy than standard clinical markers. CONCLUSIONS: This study extends the understanding of host responses underlying sepsis and COVID-19, indicating varying disease mechanisms with unique signatures. These diagnostic and severity signatures are candidates for the development of personalized management of COVID-19 and sepsis.

Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes.

BACKGROUND: Streptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase. METHODS: Bacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry. RESULTS: Here, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation. CONCLUSIONS: Our findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.

Analysis of host-pathogen gene association networks reveals patient-specific response to streptococcal and polymicrobial necrotising soft tissue infections.

BACKGROUND: Necrotising soft tissue infections (NSTIs) are rapidly progressing bacterial infections usually caused by either several pathogens in unison (polymicrobial infections) or Streptococcus pyogenes (mono-microbial infection). These infections are rare and are associated with high mortality rates. However, the underlying pathogenic mechanisms in this heterogeneous group remain elusive. METHODS: In this study, we built interactomes at both the population and individual levels consisting of host-pathogen interactions inferred from dual RNA-Seq gene transcriptomic profiles of the biopsies from NSTI patients. RESULTS: NSTI type-specific responses in the host were uncovered. The S. pyogenes mono-microbial subnetwork was enriched with host genes annotated with involved in cytokine production and regulation of response to stress. The polymicrobial network consisted of several significant associations between different species (S. pyogenes, Porphyromonas asaccharolytica and Escherichia coli) and host genes. The host genes associated with S. pyogenes in this subnetwork were characterised by cellular response to cytokines. We further found several virulence factors including hyaluronan synthase, Sic1, Isp, SagF, SagG, ScfAB-operon, Fba and genes upstream and downstream of EndoS along with bacterial housekeeping genes interacting with the human stress and immune response in various subnetworks between host and pathogen. CONCLUSIONS: At the population level, we found aetiology-dependent responses showing the potential modes of entry and immune evasion strategies employed by S. pyogenes, congruent with general cellular processes such as differentiation and proliferation. After stratifying the patients based on the subject-specific networks to study the patient-specific response, we observed different patient groups with different collagens, cytoskeleton and actin monomers in association with virulence factors, immunogenic proteins and housekeeping genes which we utilised to postulate differing modes of entry and immune evasion for different bacteria in relationship to the patients' phenotype.

The Karolinska KI/K COVID-19 Immune Atlas: An open resource for immunological research and educational purposes.

The Karolinska KI/K COVID-19 Immune Atlas project was conceptualized in March 2020 as a part of the academic research response to the developing SARS-CoV-2 pandemic. The aim was to rapidly provide a curated dataset covering the acute immune response towards SARS-CoV-2 infection in humans, as it occurred during the first wave. The Immune Atlas was built as an open resource for broad research and educational purposes. It contains a presentation of the response evoked by different immune and inflammatory cells in defined naïve patient-groups as they presented with moderate and severe COVID-19 disease. The present Resource Article describes how the Karolinska KI/K COVID-19 Immune Atlas allow scientists, students, and other interested parties to freely explore the nature of the immune response towards human SARS-CoV-2 infection in an online setting.

MAIT Cells Are Major Contributors to the Cytokine Response in Group A Streptococcal Toxic Shock Syndrome.

Streptococcal toxic shock syndrome (STSS) is a rapidly progressing, life-threatening, systemic reaction to invasive infection caused by group A streptococci (GAS). GAS superantigens are key mediators of STSS through their potent activation of T cells leading to a cytokine storm and consequently vascular leakage, shock, and multiorgan failure. Mucosal-associated invariant T (MAIT) cells recognize MR1-presented antigens derived from microbial riboflavin biosynthesis and mount protective innate-like immune responses against the microbes producing such metabolites. GAS lack de novo riboflavin synthesis, and the role of MAIT cells in STSS has therefore so far been overlooked. Here we have conducted a comprehensive analysis of human MAIT cell responses to GAS, aiming to understand the contribution of MAIT cells to the pathogenesis of STSS. We show that MAIT cells are strongly activated and represent the major T cell source of IFNγ and TNF in the early stages of response to GAS. MAIT cell activation is biphasic with a rapid TCR Vß2-specific, TNF-dominated response to superantigens and a later IL-12- and IL-18-dependent, IFNγ-dominated response to both bacterial cells and secreted factors. Depletion of MAIT cells from PBMC resulted in decreased total production of IFNγ, IL-1ß, IL-2, and TNFß. Peripheral blood MAIT cells in patients with STSS expressed elevated levels of the activation markers CD69, CD25, CD38, and HLA-DR during the acute compared with the convalescent phase. Our data demonstrate that MAIT cells are major contributors to the early cytokine response to GAS, and are therefore likely to contribute to the pathological cytokine storm underlying STSS.

Risk Factors and Predictors of Mortality in Streptococcal Necrotizing Soft-tissue Infections: A Multicenter Prospective Study.

BACKGROUND: Necrotizing soft-tissue infections (NSTI) are life-threatening conditions often caused by ß-hemolytic streptococci, group A Streptococcus (GAS) in particular. Optimal treatment is contentious. The INFECT cohort includes the largest set of prospectively enrolled streptococcal NSTI cases to date. METHODS: From the INFECT cohort of 409 adults admitted with NSTI to 5 clinical centers in Scandinavia, patients culture-positive for GAS or Streptococcus dysgalactiae (SD) were selected. Risk factors were identified by comparison with a cohort of nonnecrotizing streptococcal cellulitis. The impact of baseline factors and treatment on 90-day mortality was explored using Lasso regression. Whole-genome sequencing of bacterial isolates was used for emm typing and virulence gene profiling. RESULTS: The 126 GAS NSTI cases and 27 cases caused by SD constituted 31% and 7% of the whole NSTI cohort, respectively. When comparing to nonnecrotizing streptococcal cellulitis, streptococcal NSTI was associated to blunt trauma, absence of preexisting skin lesions, and a lower body mass index. Septic shock was significantly more frequent in GAS (65%) compared to SD (41%) and polymicrobial, nonstreptococcal NSTI (46%). Age, male sex, septic shock, and no administration of intravenous immunoglobulin (IVIG) were among factors associated with 90-day mortality. Predominant emm types were emm1, emm3, and emm28 in GAS and stG62647 in SD. CONCLUSIONS: Streptococcal NSTI was associated with several risk factors, including blunt trauma. Septic shock was more frequent in NSTI caused by GAS than in cases due to SD. Factors associated with mortality in GAS NSTI included age, septic shock, and no administration of IVIG.

Integrated Univariate, Multivariate, and Correlation-Based Network Analyses Reveal Metabolite-Specific Effects on Bacterial Growth and Biofilm Formation in Necrotizing Soft Tissue Infections.

Necrotizing soft-tissue infections (NSTIs) have multiple causes, risk factors, anatomical locations, and pathogenic mechanisms. In patients with NSTI, circulating metabolites may serve as a substrate having impact on bacterial adaptation at the site of infection. Metabolic signatures associated with NSTI may reveal the potential to be useful as diagnostic and prognostic markers and novel targets for therapy. This study used untargeted metabolomics analyses of plasma from NSTI patients (n = 34) and healthy (noninfected) controls (n = 24) to identify the metabolic signatures and connectivity patterns among metabolites associated with NSTI. Metabolite-metabolite association networks were employed to compare the metabolic profiles of NSTI patients and noninfected surgical controls. Out of 97 metabolites detected, the abundance of 33 was significantly altered in NSTI patients. Analysis of metabolite-metabolite association networks showed a more densely connected network: specifically, 20 metabolites differentially connected between NSTI and controls. A selected set of significantly altered metabolites was tested in vitro to investigate potential influence on NSTI group A streptococcal strain growth and biofilm formation. Using chemically defined media supplemented with the selected metabolites, ornithine, ribose, urea, and glucuronic acid, revealed metabolite-specific effects on both bacterial growth and biofilm formation. This study identifies for the first time an NSTI-specific metabolic signature with implications for optimized diagnostics and therapies.

Correlation Between Immunoglobulin Dose Administered and Plasma Neutralization of Streptococcal Superantigens in Patients With Necrotizing Soft Tissue Infections.

Analyses of plasma collected pre- and postadministration of intravenous immunoglobulin (IVIG) from patients with group A Streptococcus necrotizing soft tissue infections demonstrated a negative correlation between IVIG dose and toxin-triggered T-cell proliferation (r = -.67, P < .0001). One 25-g IVIG dose was sufficient to yield plasma-neutralizing activity against streptococcal superantigens. Clinical Trials Registration. NCT01790698 and NCT02111161.

Bacterial deception of MAIT cells in a cloud of superantigen and cytokines.

The bacterium Staphylococcus aureus is an important cause of the life-threatening condition toxic shock syndrome in humans. Bacterial toxins known as superantigens (SAgs) generate this illness by acting as broad activators of a substantial fraction of all T lymphocytes, bypassing the normally highly stringent T-cell receptor antigen specificity to cause a systemic inflammatory cytokine storm in the host. In a new study, Shaler et al. found that immune cells called mucosa-associated invariant T (MAIT) cells make an unexpectedly large contribution to the SAg response in a largely T-cell receptor-independent, cytokine-driven manner. Subsequent to such activation, the MAIT cells remain unresponsive to stimulation with bacterial antigen. Thus, S. aureus hijacks MAIT cells in the cytokine storm and leaves them functionally impaired. This work provides new insight into the role of MAIT cells in antibacterial immunity and opens new avenues of investigation to understand and possibly treat bacterial toxic shock and sepsis.

The INFECT-Project: An International and Multidisciplinary Project on Necrotizing Soft Tissue Infections.

This book describes clinical and microbiologic aspects, pathogenesis, and diagnostics, related to the severe and rapidly spreading necrotizing soft tissue infections. The work has its foundation in a recently completed European Union funded FP7-project called INFECT, which during the period 2013-2018 focused on utilizing a systems medicine approach to increase our understanding of these heterogenous and complex life-threatening infections. In this chapter, the aim and scope as well as key achievements of the INFECT-project are described.

Treatment of Necrotizing Soft Tissue Infections: IVIG.

Immunoglobulins are key effector molecules in the humoral immune response. Intravenous polyspecific immunoglobulin (IVIG) is a preparation of polyclonal serum immunoglobulins, typically IgG, from thousands of donors. It has been used as adjunctive therapy in critically ill patients with severe infections, i.e. sepsis, septic shock, and necrotizing soft tissue infections. IVIG has been used for patients with severe invasive group A streptococcal infection since the early nineties and off-label use of IVIG for necrotizing soft tissue infections is common. It is also used for a variety of autoimmune, inflammatory, and immunodeficiency diseases. A meta-analysis of the clinical studies available for IVIG use in group A streptococcal toxic shock syndrome indicates a survival benefit. A blinded, placebo-controlled clinical trial (INSTINCT) assessed the effect of IVIG in 100 intensive care unit patients with necrotizing soft tissue infections, including all bacterial etiologies. The study did not demonstrate any effect on self-reported physical functioning at 6 months. In this chapter, we review the mechanisms of action of IVIG and the clinical studies that are available for necrotizing soft tissue infections as well as severe group A streptococcal infections.

Pathogenic Mechanisms of Streptococcal Necrotizing Soft Tissue Infections.

Necrotizing skin and soft tissue infections (NSTIs) are severe life-threatening and rapidly progressing infections. Beta-hemolytic streptococci, particularly S. pyogenes (group A streptococci (GAS)) but also S. dysgalactiae subsp. equisimilis (SDSE, most group G and C streptococcus), are the main causative agents of monomicrobial NSTIs and certain types, such as emm1 and emm3, are over-represented in NSTI cases. An arsenal of bacterial virulence factors contribute to disease pathogenesis, which is a complex and multifactorial process. In this chapter, we summarize data that have provided mechanistic and immuno-pathologic insight into host-pathogens interactions that contribute to tissue pathology in streptococcal NSTIs. The role of streptococcal surface associated and secreted factors contributing to the hyper-inflammatory state and immune evasion, bacterial load in the tissue and persistence strategies, including intracellular survival and biofilm formation, as well as strategies to mimic NSTIs in vitro are discussed.